US20160362703A1 - Methods of improving drought tolerance and seed production in rice - Google Patents
Methods of improving drought tolerance and seed production in rice Download PDFInfo
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- US20160362703A1 US20160362703A1 US15/246,932 US201615246932A US2016362703A1 US 20160362703 A1 US20160362703 A1 US 20160362703A1 US 201615246932 A US201615246932 A US 201615246932A US 2016362703 A1 US2016362703 A1 US 2016362703A1
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- Rice is one of the three major cereal crops and is a staple food for more than a third of the world's population. Drought is one of the major abiotic stress factors limiting crop productivity worldwide. Global climate changes may further exacerbate the drought situation in major crop-producing countries. Although irrigation may in theory solve the drought problem, it is usually not a viable option because of the cost associated with building and maintaining an effective irrigation system, as well as other issues, such as the general availability of water. Thus, alternative means for alleviating plant water stress are needed.
- stress-related genes Upon exposure of plants to drought conditions, many stress-related genes are induced and their products are thought to function as cellular protectants of stress-induced damage.
- the expression of stress-related genes is largely regulated by specific transcription factors.
- Members of the AP2, bZIP, zinc finger, and MYB families have been shown to have regulatory roles in stress responses.
- the rice and Arabidopsis genomes code for more than 1300 transcriptional regulators, accounting for about 6% of the estimated total number of genes in both cases. About 45% of these transcription factors were reported to be from plant-specific families (Riechmann et al., (2000) Science 290: 2105-2110).
- Plant modification for enhanced drought tolerance is mostly based on the manipulation of either transcription and/or signaling factors or genes that directly protect plant cells against water deficit.
- understanding the basic biochemical and molecular mechanisms for drought stress perception, transduction, response and tolerance remains a major challenge. Utilization of the knowledge on drought tolerance to generate plants that can endure under extreme water deficit condition is even a bigger challenge.
- Rice is a staple food for greater than one third of the world's population and worldwide total rice planting area is approximately 1.5 million square kilometers. In 2009 global rice production was over 670 million tons, second only to maize.
- Heterotrimeric G-proteins are key signal transduction components that couple the perception of an external signal by a G-protein coupled receptor (GPCR) to downstream effectors. More than one third of mammalian signaling pathways depend on heterotrimeric G-proteins, including vision, taste, olfaction, hormones, and neurotransmitters. G-protein coupled signaling pathways are targets of approximately half of all pharmaceuticals.
- the G-protein complex is comprised of G ⁇ , G ⁇ and G ⁇ monomeric subunits that assemble as a heterotrimer that physically associates with a GPCR. Activation of the GPCR triggers the G ⁇ subunit to exchange GDP for GTP, thus activating the G-protein. Once active the heterotrimeric complex dissociates from the GPCR and the G ⁇ subunit separates from the G ⁇ heterodimer. Both GTP-bound G ⁇ and the G ⁇ heterodimer transduce the signal to downstream effectors.
- G-proteins have been studied extensively in animals. To date, 23 G ⁇ , 6 G ⁇ and 11 G ⁇ genes have been reported in mammals (Vanderbeld and Kelly (2000) Biochem. Cell Biol. 78: 537-550). The alpha subunits are classified into four subfamilies: Gs, Gi, Gq, and G 12 . In contrast, relatively little is known about the role G-proteins play in plants. Loss-of-function mutants in the G ⁇ subunit of rice and Arabidopsis are completely viable, but show several characteristic developmental attributes.
- the rice mutant exhibits shortened internodes, rounded seeds, and partial insensitivity to gibberellin, whereas the Arabidopsis mutants have rounded leaves and altered sensitivity to a number of phytohormones (Ashikari et al. (1999) Proc. Natl. Acad. Sci. 96: 10284-10289; Fujisawa et al. (1999) Proc. Natl. Acad. Sci. 96:7575-7580; Ueguchi-Tanaka et al. (2000) Proc. Natl. Acad. Sci. 97: 11638-11643; Wang et al. (2001) Science 292: 2070-2072; (Ullah et al.
- a loss-of-function mutant in the G ⁇ subunit of Arabidopsis exhibits several defects including short, blunt fruits, rounded leaves, and shortened floral buds (Lease et al. (2001) Plant Cell 13: 2631-2641).
- G ⁇ proteins such as RGA1
- the rice dwarf mutant, d1 contains a non-functional RGA1 gene, encoding the GTP-binding ⁇ -subunit of the heterotrimeric G protein.
- Rice RGA1 encodes a 380 amino acid G ⁇ protein.
- Applicants have identified a 2 bp deletion (allele d1), which results in a frameshift mutation resulting in a predicted protein truncation after amino acid 304.
- This mutant was originally isolated as a spontaneous mutant with reduced height and shorter, erect, thicker, broad, dark green leaves, compact panicles, and short, round grains.
- Applicants have identified that a non-functional RGA1 gene or other related G-proteins can be used to create higher seed production and yield under drought conditions.
- the d1 plants also perform better at higher planting density.
- the present invention provides for compositions and methods for producing plants that have higher yield in drought conditions by manipulating the G-proteins such as the RGA1 gene.
- the present invention provides for plants, varieties, lines, and hybrids, as well as plant tissues and plant seeds that contain modified G-protein activity, particularly RGA1 activity to engineer drought tolerance and improved seed production in plants.
- the present invention provides for one or more plants whose germplasm has been modified to render a G-protein, particularly the RGA1 gene or its gene product inactive. Moreover, in further embodiments the invention relates to the offspring (e.g., F1, F2, F3, etc.) of a cross of said plant wherein the germplasm of said offspring has the same mutation as the parent plant. Therefore, embodiments of the present invention provide for plant varieties/hybrids whose germplasm contains a mutation, such that the phenotype of the plants is non-functional RGA1 gene activity in drought conditions.
- offspring e.g., F1, F2, F3, etc.
- said offspring are the result of a cross between elite lines, at least one of which contains a germplasm comprising a mutation that renders the G-protein such as RGA1 protein or gene of said plant inactive, particularly during drought.
- the present invention provides a method of improving seed development in drought conditions comprising inhibiting the activity of a G-protein, such as an RGA1 protein during drought conditions.
- the present invention provides a hybrid plant, line or variety, wherein said plant hybrid, line or variety comprises germplasm comprising one or more mutations in a G-protein encoding gene, or the RGA1 gene such that the G-protein or RGA1 protein is inactive during drought conditions.
- said plant hybrid, line or variety comprises germplasm comprising one or more mutations in a G-protein encoding gene, or the RGA1 gene such that the G-protein or RGA1 protein is inactive during drought conditions.
- This can be by use of a naturally occurring mutation such as the d1 mutant, or preferably by introduction an expression cassette to said plant designed to reduce activity of the RGA1 or other related G-protein gene either constitutively or under drought conditions.
- said plant hybrid, line or variety is created by introgression of a plant germplasm that comprises said one or more modifications for rendering a G-protein or RGA1 gene or its encoded protein product inactive.
- said plant hybrid, line or variety is created by incorporation of a heterologous genetic construct comprising designed to inhibit or otherwise decrease G-protein activity, particularly during drought conditions.
- the present invention provides a method for producing a plant hybrid, line or variety resistant to drought conditions and with improved seed production compared to a wild type plant under the same conditions comprising identifying a germplasm with a null G-protein or RGA1 modification, and introducing said germplasm into an elite plant hybrid, line or variety.
- said introducing of said germplasm into said elite hybrid, line or variety is by introgression.
- said introducing of said germplasm into said elite plant hybrid, line or variety is by introduction of a heterologous genetic construct.
- the present invention provides a plant hybrid, line or variety wherein the germplasm of said hybrid, line or variety confers drought tolerance with improved seed production.
- the present invention provides a method for identifying plant lines resistant to drought comprising supplying a nucleic acid sample from a plant, providing amplification primers for amplifying a region of a plant corresponding to an G-protein or RGA1 nucleic acid sample, applying said amplification primers to said nucleic acid sample such that amplification of said region of said G-protein gene occurs, and identifying plants resistant to drought based on the presence of one or more null mutations in said amplified nucleic acid sample.
- the present invention provides for seeds wherein said germplasm of said seeds comprises a modified G-protein or RGA1 gene such that said mutation confers improved seed production under drought conditions.
- the present invention provides for a plant that comprises a heterologous nucleotide sequence that is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the RGA1 gene of SEQ ID NO: 1, 2, 5, or 6.
- the invention also includes RGA1 polypeptides including the amino acid sequences of SEQ ID NO:3 or 4, conservatively modified variants which retain activity or loss of activity thereof as applicable.
- the present invention provides a method of producing plant seed comprising crossing a plant comprising a genetically modified G-protein or a genetically modified RGA1 gene with itself or a second plant and collecting said seed from said cross.
- the methods for producing said seed comprises planting a parent seed line wherein said parent seed line comprises a germplasm confers a decrease in activity or a G-protein or RGA1 mutation with a parent pollinator plant line wherein said pollinator and/or seed line germplasm comprises a germplasm that confers a modified G-protein or RGA1 protein, growing said parent seed and pollinator plants together, allowing for the said parent seed plants to be pollinated by said parent pollinator plants, and harvesting the seed that results from said pollination.
- the invention provides for genetically modified plants incorporating a heterologous nucleotide construct encoding a RGA1 gene such as SEQ ID NOS: 1, 2, 5, or 6 operably linked to regulatory sequences such as expression cassettes, inhibition constructs, plants, plant cells, and seeds.
- the genetically modified plants, plant cells, and seeds of the invention may exhibit phenotypic changes, such as modulated RGA1 activity, particularly during drought conditions.
- Methods are provided for reducing or eliminating the activity of a G-protein or RGA1 polypeptide in a plant, comprising introducing into the plant a selected polynucleotide.
- providing the polynucleotide decreases the level of RGA1 or related G-protein in the plant.
- Methods are also provided for increasing the level of a modified RGA1 or G-protein polypeptide in a plant either constitutively or at specifically regulated times and tissues comprising introducing into the plant a selected polynucleotide with appropriate regulatory elements.
- expression of the modified RGA1 or G-protein polynucleotide improves the plants' tolerance to drought.
- nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
- Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range.
- Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission.
- Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
- software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th Edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
- amplified is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template.
- Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Canteen, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications , D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
- chromosomal region includes reference to a length of a chromosome that may be measured by reference to the linear segment of DNA that it comprises.
- the chromosomal region can be defined by reference to two unique DNA sequences, i.e., markers.
- “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
- nucleic acid variations are “silent variations” and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid.
- each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine; and UGG, which is ordinarily the only codon for tryptophan
- each silent variation of a nucleic acid which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and is within the scope of the present invention.
- amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
- any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered.
- 1, 2, 3, 4, 5, 7, or 10 alterations can be made.
- Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived.
- substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for its native substrate.
- Conservative substitution tables providing functionally similar amino acids are well known in the art.
- nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA).
- the information by which a protein is encoded is specified by the use of codons.
- the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code.
- variants of the universal code such as are present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum , or the ciliate Macronucleus, may be used when the nucleic acid is expressed therein.
- nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)).
- full-length sequence in reference to a specified polynucleotide or its encoded protein means having the entire amino acid sequence of, a native (non-synthetic), endogenous, biologically active form of the specified protein.
- Methods to determine whether a sequence is full-length are well known in the art including such exemplary techniques as northern or western blots, primer extensions, S1 protection, and ribonuclease protection. See, e.g., Plant Molecular Biology: A Laboratory Manual , Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to known full-length homologous (orthologous and/or paralogous) sequences can also be used to identify full-length sequences of the present invention.
- consensus sequences typically present at the 5′ and 3′ untranslated regions of mRNA aid in the identification of a polynucleotide as full-length.
- the consensus sequence ANNNN AUG G where the underlined codon represents the N-terminal methionine, aids in determining whether the polynucleotide has a complete 5′ end.
- Consensus sequences at the 3′ end such as polyadenylation sequences, aid in determining whether the polynucleotide has a complete 3′ end.
- heterologous in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
- a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form.
- a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
- host cell is meant a cell which contains a vector and supports the replication and/or expression of the vector.
- Host cells may be prokaryotic cells such as E. coli , or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells.
- host cells are monocotyledonous or dicotyledonous plant cells.
- a particularly preferred monocotyledonous host cell is a maize host cell.
- hybridization complex includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
- the term “introduced” in the context of inserting a nucleic acid into a cell means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
- isolated refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment.
- the isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment.
- the alteration to yield the synthetic material can be performed on the material within or removed from its natural state.
- a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by means of human intervention performed within the cell from which it originates. See, e.g., Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868.
- a naturally occurring nucleic acid e.g., a promoter
- Nucleic acids which are “isolated” as defined herein are also referred to as “heterologous” nucleic acids.
- chromosomal region defined by and including with respect to particular markers includes reference to a contiguous length of a chromosome delimited by and including the stated markers.
- marker includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome.
- a “polymorphic marker” includes reference to a marker which appears in multiple forms (alleles) such that different forms of the marker, when they are present in a homologous pair, allow transmission of each of the chromosomes of that pair to be followed.
- a genotype may be defined by use of one or a plurality of markers.
- nucleic acid library is meant a collection of isolated DNA or cDNA molecules which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology , Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology , F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994).
- operably linked includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence.
- operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
- plant can include reference to whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells, seeds and progeny of same.
- Plant cell as used herein, further includes, without limitation, cells obtained from or found in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues.
- the class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Particularly preferred plants include maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.
- polynucleotide includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s).
- a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
- DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
- polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
- polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
- the terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
- the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
- polypeptide “peptide” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides are not entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally.
- Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Further, this invention contemplates the use of both the methionine-containing and the methionine-less amino terminal variants of the protein of the invention.
- promoter includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
- a “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium . Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissue are referred to as “tissue specific”.
- a “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves.
- An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters.
- a “constitutive” promoter is a promoter which is active under most environmental conditions.
- recombinant includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified.
- recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention.
- the term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
- a “recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell.
- the recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment.
- the recombinant expression cassette portion of an expression vector includes, among other sequences, nucleic acids to be transcribed, and a promoter.
- amino acid residue or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”).
- the amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
- sequences include reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.
- Selectively hybridizing sequences typically have about at least 80% sequence identity, preferably 90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.
- stringent conditions or “stringent hybridization conditions” includes reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and can be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
- stringent conditions will be those in which the salt concentration is less than about 1.5 M Na + ion, typically about 0.01 to 1.0 M Na + ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides).
- Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
- Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5 ⁇ to 1 ⁇ SSC at 55 to 50° C.
- Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1 ⁇ SSC at 60 to 65° C. for 20 minutes.
- T m 81.5° C.+16.6 (log M)+0.41 (% GC) ⁇ 0.61 (% form) ⁇ 500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
- the T m is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. T m is reduced by about 1° C. for each 1% of mismatching; thus, T m , hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with 90% identity are sought, the T m can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T m ) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C.
- T m thermal melting point
- moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T m ); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (T m ).
- T m thermal melting point
- heterologous polynucleotide As used herein, “genetically modified plant” includes reference to a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
- Transgenic is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
- transgenic does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
- vector includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
- sequence relationships between two or more nucleic acids or polynucleotides are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
- reference sequence is a defined sequence used as a basis for sequence comparison.
- a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
- comparison window includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
- the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer.
- Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci.
- the BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences.
- sequence identity/similarity values refer to the value obtained using the BLAST 2.0 suite of programs using default parameters.
- Altschul et al. Nucleic Acids Res. 25:3389-3402 (1997).
- Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information World Wide Web at ncbi.nih.gov.
- This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra).
- initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
- the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
- the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
- the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
- the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)).
- One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
- BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar.
- a number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.
- sequence identity in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window.
- sequence identity When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have “sequence similarity” or “similarity”.
- Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1.
- the scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
- percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
- polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters.
- sequence identity preferably at least 80%, more preferably at least 90% and most preferably at least 95%.
- nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
- One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid sequence encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid sequence.
- substantially identical in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, or preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window.
- optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970).
- An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide.
- a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution.
- Peptides which are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
- FIG. 1 shows the effects of water stress on wild-type and d1 plants during flowering and grain filling; mutant plants remain dark green and healthy while wild-type plants dry out and have a few living leaves under severe drought.
- the three watering conditions were: well-watered (100% soil relative water content), medium-water (45% soil relative water content), and low water (30% soil relative water content) conditions. Photographs were taken 140-160 days after emergence.
- FIG. 2 shows leaf temperatures on plants 165 days after emergence were measured by infrared thermography using a FLIR T620 Thermal Imaging Camera (FLIR Systems, USA). In order to obtain reliable comparisons, images were obtained over the shortest time interval possible; approximately 15 minutes total. Pseudo-colored temperature scales are indicated directly on the photographs.
- FIG. 3 shows that net photosynthesis and stomatal conductance measured on leaves between 120 and 130 days after emergence, using a Li-Cor 6400 Portable Photosynthesis System. Light intensity was 500 ⁇ mol m ⁇ 2 s ⁇ 1 .
- FIG. 4 shows the relationship between stomatal conductance and transpiration, measured under ambient conditions of 500 ⁇ mol m ⁇ 2 s ⁇ 1 light intensity and 30° C. temperature using a Li-Cor 6400 Portable Photosynthesis System. Measurements were taken on seedlings at 40 days after emergence. Each data point represents an individual plant. Lines depicted are regression lines for each genotype; ANCOVA analysis demonstrated significant differences (P ⁇ 0.05) in the slopes.
- FIG. 5 shows ⁇ leaf measurements, which were made using a Scholander pressure chamber. Measurements were taken on the flag leaf of the primary tiller of plants 120-130 days after emergence.
- FIG. 6 is a graph showing the percentage of stalks flowering at the end of the reproductive phase in high water, medium water, and low water conditions.
- FIG. 7 shows the average number of panicles produced through time in d1 and wild-type plants.
- FIG. 8 shows the percentage of grain viability at the end of the reproductive cycle under high water, medium water and low water conditions. Data were obtained from 6 panicles randomly selected for analysis from 6 different plants of each genotype and water treatment combination, measured at the end of the life cycle.
- FIG. 9 is graph of yield of d1 and WT in well watered, medium water and low water conditions.
- FIG. 10 shows net photosynthesis and stomatal conductance at 1500 ⁇ mol m ⁇ 2 s ⁇ 1 light measured on the flag leaf of the primary tiller at 60 days after emergence using a Li-Cor 6400 Portable Photosynthesis System.
- FIG. 11 shows that high planting density compromises plant growth and survival (as indicated by plant height) earlier in wild-type than in d1 plants.
- FIGS. 12A-B is a sequence alignment of the coding region of rgal and the d1 allele. (SEQ ID NO: 1 and 2 respectively).
- FIG. 13 is a sequence alignment of the encoded proteins of rgal and the d1 allele. (SEQ ID NO: 3 and 4 respectively).
- FIGS. 14A-D is a sequence alignment of the genomic region of rgal and the d1 allele. (SEQ ID NO: 5 and 6 respectively).
- the rice dwarf mutant, d1 contains a non-functional RGA1 gene, encoding the GTP-binding ⁇ -subunit of the heterotrimeric G protein.
- This mutant was originally isolated as a spontaneous mutant with reduced height and shorter, erect, thicker, broad, dark green leaves, compact panicles, and short, round grains. Applicants have identified that a non-functional RGA1 gene or other related G-proteins can be used to create higher seed production and yield under drought conditions.
- the present invention provides for compositions and methods for producing plants that have higher yield in drought conditions by manipulating the G-proteins such as the RGA1 gene or protein.
- the present invention provides for plants, varieties, lines, and hybrids, as well as plant tissues and plant seeds that contain modified G-protein activity, particularly RGA1 activity to engineer drought tolerance and improved seed production in plants.
- the present invention provides for one or more plants whose germplasm has been modified to render a G-protein, particularly the RGA1 gene or its gene product inactive. Moreover, in further embodiments the invention relates to the offspring (e.g., F1, F2, F3, etc.) of a cross of said plant wherein the germplasm of said offspring has the same mutation as the parent plant. Therefore, embodiments of the present invention provide for plant varieties/hybrids whose germplasm contains a mutation, such that the phenotype of the plants is non-functional RGA1 gene activity in drought conditions.
- offspring e.g., F1, F2, F3, etc.
- said offspring are the result of a cross between elite lines, at least one of which contains a germplasm comprising a mutation that renders the G-protein such as RGA1 protein or gene of said plant inactive, particularly during drought.
- the present invention provides a method of improving seed development in drought conditions comprising inhibiting the activity of a G-protein, such as RGA1 protein during drought conditions.
- the present invention provides a hybrid plant, line or variety, wherein said plant hybrid, line or variety comprises germplasm comprising one or more mutations in a G-protein encoding gene, or the RGA1 gene such that the G-protein or RGA1 protein is inactive during drought conditions.
- said plant hybrid, line or variety comprises germplasm comprising one or more mutations in a G-protein encoding gene, or the RGA1 gene such that the G-protein or RGA1 protein is inactive during drought conditions.
- This can be by use of a naturally occurring mutation such as the d1 mutant, or preferably by introduction an expression cassette to said plant designed to reduce activity of the RGA1 or other related G-protein gene either constitutively or under drought conditions.
- said plant hybrid, line or variety is created by introgression of a plant germplasm that comprises said one or more modifications for rendering a G-protein or RGA1 gene or its encoded protein product inactive.
- said plant hybrid, line or variety is created by incorporation of a heterologous genetic construct comprising designed to inhibit or otherwise decrease G-protein activity, particularly during drought conditions.
- the present invention provides a method for producing a plant hybrid, line or variety resistant to drought conditions and with improved seed production compared to a wild type plant under the same conditions comprising identifying a germplasm with a null G-protein or RGA1 modification, and introducing said germplasm into an elite plant hybrid, line or variety.
- said introducing of said germplasm into said elite hybrid, line or variety is by introgression.
- said introducing of said germplasm into said elite plant hybrid, line or variety is by introduction of a heterologous genetic construct.
- the present invention provides a plant hybrid, line or variety wherein the germplasm of said hybrid, line or variety comprises drought tolerance with improved seed production.
- the present invention provides a method for identifying plant lines resistant to drought comprising supplying a nucleic acid sample from a plant, providing amplification primers for amplifying a region of a plant corresponding to a G-protein or RGA1 nucleic acid sample, applying said amplification primers to said nucleic acid sample such that amplification of said region of said G-protein gene occurs, and identifying plants resistant to drought based on the presence of one or more null mutations in said amplified nucleic acid sample.
- the present invention provides for seeds wherein said germplasm of said seeds comprises a modified G-protein or RGA1 gene such that said mutation confers improved seed production under drought conditions.
- the present invention provides for a plant that comprises a heterologous nucleotide sequence that is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the RGA1 gene of SEQ ID NO:1, 2, 5, or 6.
- the invention also includes RGA1 polypeptides including the amino acid sequences of SEQ ID NO: 3 or 4, conservatively modified variants which retain activity or loss of activity thereof as applicable.
- the present invention provides a method of producing plant seed comprising crossing a plant comprising a genetically modified G-protein or RGA1 gene with itself or a second plant and collecting said seed from said cross.
- the methods for producing said seed comprises planting a parent seed line wherein said parent seed line comprises a germplasm confers a decrease in activity or a G-protein or RGA1 mutation with a parent pollinator plant line wherein said pollinator and/or seed line germplasm comprises a germplasm that confers a modified G-protein of RGA1 protein, growing said parent seed and pollinator plants together, allowing for the said parent seed plants to be pollinated by said parent pollinator plants, and harvesting the seed that results from said pollination.
- the invention provides for genetically modified plants incorporating a heterologous nucleotide construct encoding a RGA1 gene such as SEQ ID NOS: 1, 2, 5, or 6 operably linked to regulatory sequences such as expression cassettes, inhibition constructs, plants, plant cells, and seeds.
- the genetically modified plants, plant cells, and seeds of the invention may exhibit phenotypic changes, such as modulated RGA1 activity, particularly during drought conditions.
- Methods are provided for reducing or eliminating the activity of a G-protein or RGA1 polypeptide in a plant, comprising introducing into the plant a selected polynucleotide.
- providing the polynucleotide decreases the level of RGA1 or related G-protein in the plant.
- Methods are also provided for increasing the level of a modified RGA1 or G-protein polypeptide in a plant either constitutively or at specifically regulated times and tissues comprising introducing into the plant a selected polynucleotide with appropriate regulatory elements.
- expression of the modified RGA1 or G-protein polynucleotide improves the plant's tolerance to drought.
- constructs, promoters or control systems used in the methods of the invention may include a tissue specific promoter, an inducible promoter or a constitutive promoter.
- CaMV cauliflower mosaic virus 35S. It has been shown to be highly active in many plant organs and during many stages of development when integrated into the genome of transgenic plants and has been shown to confer expression in protoplasts of both dicots and monocots.
- Organ-specific promoters are also well known.
- the E8 promoter is only transcriptionally activated during tomato fruit ripening, and can be used to target gene expression in ripening tomato fruit (Deikman and Fischer, EMBO J . (1988) 7:3315; Giovannoni et al., The Plant Cell (1989) 1:53).
- the activity of the E8 promoter is not limited to tomato fruit, but is thought to be compatible with any system wherein ethylene activates biological processes.
- the Lipoxegenase (“the LOX gene”) is a fruit specific promoter.
- Leaf specific promoters include as the AS-1 promoter disclosed in U.S. Pat. No. 5,256,558 to Coruzzi and the RBCS-3A promoter isolated from pea the RBCS-3A gene disclosed in U.S. Pat. No. 5,023,179 to Lam et al.
- root specific promoters include the CamV 35S promoter disclosed in U.S. Pat. No. 391,725 to Coruzzi et al; the RB7 promoter disclosed in U.S. Pat. No. 5,459,252 to Conking et al. and the promoter isolated from Brassica napus disclosed in U.S. Pat. No. 5,401,836 to Bazczynski et al. which give root specific expression.
- promoters include maternal tissue promoters such as seed coat, pericarp and ovule. Promoters highly expressed early in endosperm development are most effective in this application. Of particular interest is the promoter from the a′ subunit of the soybean ⁇ -conglycinin gene [Walling et al., Proc. Natl. Acad. Sci. USA 83:2123-2127 (1986)] which is expressed early in seed development in the endosperm and the embryo.
- Further seed specific promoters include the Napin promoter described in U.S. Pat. No. 5,110,728 to Calgene, which describes and discloses the use of the napin promoter in directing the expression to seed tissue of an acyl carrier protein to enhance seed oil production; the DC3 promoter from carrots which is early to mid embryo specific and is disclosed at Plant Physiology , October 1992 100(2) p. 576-581, “Hormonal and Environmental Regulation of the Carrot Lea-class Gene Dc 3, and Plant Mol. Biol ., April 1992, 18(6) p. 1049-1063, “Transcriptional Regulation of a Seed Specific Carrot Gene, DC 8”: the phaseolin promoter described in U.S. Pat. No. 5,504,200 to Mycogen which discloses the gene sequence and regulatory regions for phaseolin, a protein isolated from P. vulgaris which is expressed only while the seed is developing within the pod, and only in tissues involved in seed generation.
- organ-specific promoters appropriate for a desired target organ can be isolated using known procedures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, Phil, Trans. R. Soc. London (1986) B314-343. mRNAs are first isolated to obtain suitable probes for retrieval of the appropriate genomic sequence which retains the presence of the natively associated control sequences. An example of the use of techniques to obtain the cDNA associated with mRNA specific to avocado fruit is found in Christoffersen et al., Plant Molecular Biology (1984) 3:385. Briefly, mRNA was isolated from ripening avocado fruit and used to make a cDNA library.
- Clones in the library were identified that hybridized with labeled RNA isolated from ripening avocado fruit, but that did not hybridize with labeled RNAs isolated from unripe avocado fruit. Many of these clones represent mRNAs encoded by genes that are transcriptionally activated at the onset of avocado fruit ripening.
- the promoter used in the method of the invention may be an inducible promoter.
- An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of a DNA sequence in response to an inducer. In the absence of an inducer, the DNA sequence will not be transcribed.
- the protein factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer.
- the inducer may be a chemical agent such as a protein, metabolite (sugar, alcohol etc.), a growth regulator, herbicide, or a phenolic compound or a physiological stress imposed directly by heat, salt, toxic elements etc.
- a plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell such as by spraying, watering, heating, or similar methods.
- inducible promoters include the inducible 70 kD heat shock promoter of (Freeling, M., Bennet, D. C., Maize ADN 1 , Ann. Rev. of Genetics, 19:297-323) and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, R. T., et al., Miflin, B. J., Ed. Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3, p.
- the inducible promoter may be in an induced state throughout seed formation or at least for a period which corresponds to the transcription of the DNA sequence of the recombinant DNA molecule(s).
- an inducible promoter is the chemically inducible gene promoter sequence isolated from a 27 kD subunit of the maize glutathione-S-transferase (GST II) gene.
- a number of other potential inducers may be used with this promoter as described in published PCT Application No. PCT/GB90/00110 by ICI.
- inducible promoter is the light inducible chlorophyll a/b binding protein (CAB) promoter, also described in published PCT Application No. PCT/GB90/00110 by ICI.
- CAB chlorophyll a/b binding protein
- inducible promoters have also been described in published Application No. EP89/103888.7 by Ciba-Geigy.
- PR protein genes especially the tobacco PR protein genes, such as PR-1a, PR-1b, PR-1c, PR-1, PR-A, PR-S, the cucumber chitinase gene, and the acidic and basic tobacco beta-1,3-glucanase genes.
- inducers for these promoters as described in Application No. EP89/103888.7.
- the preferred promoters may be used in conjunction with naturally occurring flanking coding or transcribed sequences of the feronia regulatory genes or with any other coding or transcribed sequence that is critical to pollen tube formation and/or fertilization.
- intron sequences may also be desirable to include some intron sequences in the promoter constructs since the inclusion of intron sequences in the coding region may result in enhanced expression and specificity.
- regions of one promoter may be joined to regions from a different promoter in order to obtain the desired promoter activity resulting in a chimeric promoter.
- Synthetic promoters which regulate gene expression may also be used.
- the expression system may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer elements.
- an expression cassette or construct should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
- the termination region or polyadenylation signal may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
- Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., EMBO J . (1984) 3:835-846) or the nopaline synthase signal (Depicker et al., Mol. and Appl. Genet . (1982) 1:561-573).
- Recombinant DNA molecules containing any of the DNA sequences and promoters described herein may additionally contain selection marker genes which encode a selection gene product which confer on a plant cell resistance to a chemical agent or physiological stress, or confers a distinguishable phenotypic characteristic to the cells such that plant cells transformed with the recombinant DNA molecule may be easily selected using a selective agent.
- selection marker gene is neomycin phosphotransferase (NPT II) which confers resistance to kanamycin and the antibiotic G-418.
- Cells transformed with this selection marker gene may be selected for by assaying for the presence in vitro of phosphorylation of kanamycin using techniques described in the literature or by testing for the presence of the mRNA coding for the NPT II gene by Northern blot analysis in RNA from the tissue of the transformed plant. Polymerase chain reactions are also used to identify the presence of a transgene or expression using reverse transcriptase PCR amplification to monitor expression and PCR on genomic DNA. Other commonly used selection markers include the ampicillin resistance gene, the tetracycline resistance and the hygromycin resistance gene. Transformed plant cells thus selected can be induced to differentiate into plant structures which will eventually yield whole plants. It is to be understood that a selection marker gene may also be native to a plant.
- A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells.
- the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant. Sci. 10: 1 (1991).
- a generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 ⁇ m.
- the expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes.
- selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.
- a plant cell be transformed with a recombinant DNA molecule containing at least two DNA sequences or be transformed with more than one recombinant DNA molecule.
- the DNA sequences or recombinant DNA molecules in such embodiments may be physically linked, by being in the same vector, or physically separate on different vectors.
- a cell may be simultaneously transformed with more than one vector provided that each vector has a unique selection marker gene.
- a cell may be transformed with more than one vector sequentially allowing an intermediate regeneration step after transformation with the first vector.
- it may be possible to perform a sexual cross between individual plants or plant lines containing different DNA sequences or recombinant DNA molecules preferably the DNA sequences or the recombinant molecules are linked or located on the same chromosome, and then selecting from the progeny of the cross, plants containing both DNA sequences or recombinant DNA molecules.
- Expression of recombinant DNA molecules containing the DNA sequences and promoters described herein in transformed plant cells may be monitored using Northern blot techniques and/or Southern blot techniques known to those of skill in the art.
- the transformed cells may then be regenerated into a transgenic plant.
- the regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner.
- the expression or inhibition cassette After the expression or inhibition cassette is stably incorporated into regenerated transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. It may be useful to generate a number of individual transformed plants with any recombinant construct in order to recover plants free from any position effects. It may also be preferable to select plants that contain more than one copy of the introduced recombinant DNA molecule such that high levels of expression of the recombinant molecule are obtained.
- plants may be self-fertilized, leading to the production of a mixture of seed that consists of, in the simplest case, three types, homozygous (25%), heterozygous (50%) and null (25%) for the inserted gene.
- homozygous 25%
- heterozygous 50%)
- null 25%)
- Transgenic homozygous parental lines make possible the production of hybrid plants and seeds which will contain a modified protein component.
- Transgenic homozygous parental lines are maintained with each parent containing either the first or second recombinant DNA sequence operably linked to a promoter. Also incorporated in this scheme are the advantages of growing a hybrid crop, including the combining of more valuable traits and hybrid vigor.
- nucleotide constructs of the invention also encompass nucleotide constructs that may be employed in methods for altering or mutating a genomic nucleotide sequence in an organism, including, but not limited to, chimeric vectors, chimeric mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides, self-complementary chimeric oligonucleotides, and recombinogenic oligonucleobases.
- nucleotide constructs and methods of use such as, for example, chimeraplasty, are known in the art.
- Chimeraplasty involves the use of such nucleotide constructs to introduce site-specific changes into the sequence of genomic DNA within an organism. See, U.S. Pat.
- the present invention provides a method of genotyping a plant comprising a heterologous polynucleotide of the present invention.
- Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population.
- Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. See, e.g., Clark, Ed., Plant Molecular Biology: A Laboratory Manual. Berlin, Springer Verlag, 1997. Chapter 7.
- For molecular marker methods see generally, “The DNA Revolution” in: Paterson, A. H., Genome Mapping in Plants (Austin, Tex., Academic Press/R. G. Landis Company, 1996) pp. 7-21.
- RFLPs restriction fragment length polymorphisms
- RFLPs are the product of allelic differences between DNA restriction fragments resulting from nucleotide sequence variability.
- RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction enzyme. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred. Restriction fragments from homologous chromosomes are revealed.
- the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis.
- Linked chromosomal sequences are within 50 centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of a gene of the present invention.
- the nucleic acid probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective hybridization conditions, to a gene encoding a polynucleotide of the present invention.
- the probes are selected from polynucleotides of the present invention.
- these probes are cDNA probes or restriction-enzyme treated (e.g., Pst I) genomic clones.
- the length of the probes is discussed in greater detail, supra, but are typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length.
- the probes are single copy probes that hybridize to a unique locus in a haploid chromosome complement.
- Some exemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv, and SstI.
- restriction enzyme includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves at a specific nucleotide sequence.
- the method of detecting an RFLP comprises the steps of (a) digesting genomic DNA of a plant with a restriction enzyme; (b) hybridizing a nucleic acid probe, under selective hybridization conditions, to a sequence of a polynucleotide of the present of said genomic DNA; (c) detecting therefrom a RFLP.
- polymorphic (allelic) variants of polynucleotides of the present invention can be had by utilizing molecular marker techniques well known to those of skill in the art including such techniques as: 1) single stranded conformation analysis (SSCA); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6) allele-specific PCR.
- molecular marker techniques well known to those of skill in the art including such techniques as: 1) single stranded conformation analysis (SSCA); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6) allele
- the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe.
- a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe.
- the sample is a plant sample; preferably, a sample suspected of comprising a maize polynucleotide of the present invention (e.g., gene, mRNA).
- the nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymorphic marker. Selective hybridization of the nucleic acid probe to the polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample.
- the nucleic acid probe comprises a polynucleotide of the present invention.
- RNA Ribonucleic Acids Res. 15: 8125 (1987)
- Negative elements include stable intramolecular 5′ UTR stem-loop structures (Muesing et al., Cell 48: 691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5 ‘UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8: 284 (1988)). Accordingly, the present invention provides 5’ and/or 3′ untranslated regions for modulation of translation of heterologous coding sequences.
- polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage.
- Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host such as to optimize the codon usage in a heterologous sequence for expression in maize.
- Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as “Codon Preference” available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.).
- the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention.
- the number of polynucleotides that can be used to determine a codon usage frequency can be any integer from 1 to the number of polynucleotides of the present invention as provided herein.
- the polynucleotides will be full-length sequences.
- An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50, or 100.
- sequence shuffling provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT publication No. WO 97/20078. See also, Zhang, J.-H., et al. Proc. Natl. Acad. Sci. USA 94: 4504-4509 (1997). Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo.
- the population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method.
- the characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property.
- the selected characteristic will be a decreased Km and/or increased KCat over the wild-type protein as provided herein.
- a protein or polynucleotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide.
- the increase in such properties can be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value.
- Polynucleotides and polypeptides of the present invention further include those having: (a) a generic sequence of at least two homologous polynucleotides or polypeptides, respectively, of the present invention; and, (b) a consensus sequence of at least three homologous polynucleotides or polypeptides, respectively, of the present invention.
- the generic sequence of the present invention comprises each species of polypeptide or polynucleotide embraced by the generic polypeptide or polynucleotide sequence, respectively.
- the individual species encompassed by a polynucleotide having an amino acid or nucleic acid consensus sequence can be used to generate antibodies or produce nucleic acid probes or primers to screen for homologs in other species, genera, families, orders, classes, phyla, or kingdoms.
- a polynucleotide having a consensus sequence generated from orthologous genes can be used to identify or isolate orthologs of other taxa.
- a polynucleotide having a consensus sequence will be at least 25, 30, or 40 in nucleotides in length.
- a conservative amino acid substitution can be used for amino acids which differ amongst aligned sequences but are from the same conservative substitution group as discussed above.
- no more than 1 or 2 conservative amino acids are substituted for each 10 amino acid length of consensus sequence.
- Similar sequences used for generation of a consensus or generic sequence include any number and combination of allelic variants of the same gene, orthologous, or paralogous sequences as provided herein.
- similar sequences used in generating a consensus or generic sequence are identified using the BLAST algorithm's smallest sum probability (P(N)).
- P(N) BLAST algorithm's smallest sum probability
- a polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, or 0.001, and most preferably less than about 0.0001, or 0.00001.
- Similar polynucleotides can be aligned and a consensus or generic sequence generated using multiple sequence alignment software available from a number of commercial suppliers such as the Genetics Computer Group's (Madison, Wis.) PILEUP software, Vector NTI's (North Bethesda, Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.) SEQUENCHER. Conveniently, default parameters of such software can be used to generate consensus or generic sequences.
- Methods are also provided to reduce or eliminate the activity of an RGA1 polypeptide by transforming a plant cell with an expression cassette that expresses a polynucleotide that inhibits the expression of the RGA1.
- the polynucleotide may inhibit the expression of the RGA1 directly, by preventing transcription or translation of the RGA1 messenger RNA, or indirectly, by encoding a polypeptide that inhibits the transcription or translation of an RGA1 gene encoding an RGA1 polypeptide.
- Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of the RGA1 polypeptide. Many methods may be used to reduce or eliminate the activity of an RGA1 polypeptide. In addition, more than one method may be used to reduce the activity of a single RGA1 polypeptide.
- a plant is transformed with an expression cassette that is capable of expressing a polynucleotide that inhibits the expression of an RGA1 polypeptide of the invention.
- expression refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product.
- an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one RGA1 polypeptide is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one RGA1 polypeptide of the invention.
- the “expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide
- the “expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.
- polynucleotides that inhibit the expression of an RGA1 polypeptide include sense Suppression/Cosuppression, where an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding an RGA1 polypeptide in the “sense” orientation and over expression of the RNA molecule can result in reduced expression of the native gene; Antisense Suppression where the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the RGA1 polypeptide and over expression of the antisense RNA molecule can result in reduced expression of the native gene; Double-Stranded RNA Interference, where a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA, Hairpin RNA Interference and Intron-Containing Hairpin RNA Interference.
- the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem, Small Interfering RNA or Micro RNA, where the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA such as small interfering RNA or artificial micro RNAs.
- the polynucleotide encodes a zinc finger protein that binds to a gene encoding an RGA1 polypeptide, resulting in reduced expression of the gene methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242, and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in U.S. Patent Publication No. 2003/0037355; each of which is herein incorporated by reference.
- the polynucleotide encodes an antibody that binds to at least one RGA1 polypeptide and reduces the activity of the RGA1 polypeptide.
- the expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36, incorporated herein by reference.
- the activity of an RGA1 polypeptide is reduced or eliminated by disrupting the gene encoding the RGA1 polypeptide.
- the gene encoding the RGA1 polypeptide may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing plants using random or targeted mutagenesis, and selecting for plants that have a desired trait or phenotype.
- the nucleic acid sequences of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype.
- the polynucleotides of the present invention may be stacked with any other polynucleotides of the present invention, (SEQ ID NO: 1), or with other genes implicated in herbicide resistance.
- the combinations generated can also include multiple copies of any one of the polynucleotides of interest.
- the polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No.
- the polynucleotides of the present invention can also be stacked with traits desirable for insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al (1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al.
- traits desirable for insect, disease or herbicide resistance e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et
- acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations
- inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene and GAT gene)
- traits desirable for processing or process products such as high oil (U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
- modified starches e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)
- AGPase ADPG pyrophosphorylases
- SS starch synthases
- SBE starch branching enzymes
- SDBE starch debranching enzymes
- polymers or bioplastics U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847), which facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference.
- polynucleotides of the present invention could also combine with polynucleotides providing agronomic traits such as male sterility (see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (see, WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.
- agronomic traits such as male sterility (see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (see, WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.
- stacked combinations can be created by any method including, but not limited to, polynucleotide sequences of interest can be combined at any time and in any order.
- a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation.
- the traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes.
- the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters.
- the transformed plants of the invention may be used in a plant breeding program.
- the goal of plant breeding is to combine, in a single variety or hybrid, various desirable traits.
- these traits may include, for example, resistance to diseases and insects, tolerance to heat and drought, tolerance to high planting density, reduced time to crop maturity, greater yield, and better agronomic quality.
- uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant height is desirable.
- Traditional plant breeding is an important tool in developing new and improved commercial crops.
- This invention encompasses methods for producing a plant by crossing a first parent plant with a second parent plant wherein one or both of the parent plants is a transformed plant according to the invention displaying drought tolerance as described herein.
- Plant breeding techniques known in the art and used in a plant breeding program include, but are not limited to, recurrent selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, doubled haploids, and transformation. Often combinations of these techniques are used.
- a genetic trait which has been engineered into a particular plant using transformation techniques can be moved into another line using traditional breeding techniques that are well known in the plant breeding arts. For example, a backcrossing approach is commonly used to move a transgene from a transformed maize plant to an elite inbred line, and the resulting progeny would then comprise the transgene(s). Also, if an inbred line was used for the transformation, then the transgenic plants could be crossed to a different inbred in order to produce a transgenic hybrid plant. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
- the development of a hybrid in a plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, while different from each other, breed true and are highly uniform; and (3) crossing the selected inbred lines with different inbred lines to produce the hybrids.
- the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid.
- An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a defined pair of inbreds will always be the same.
- Transgenic plants of the present invention may be used to produce, e.g., a single cross hybrid, a three-way hybrid or a double cross hybrid.
- a single cross hybrid is produced when two inbred lines are crossed to produce the F1 progeny.
- a double cross hybrid is produced from four inbred lines crossed in pairs (A ⁇ B and C ⁇ D) and then the two F1 hybrids are crossed again (A ⁇ B) ⁇ (C ⁇ D).
- a three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A ⁇ B) and then the resulting F1 hybrid is crossed with the third inbred (A ⁇ B) ⁇ C.
- Much of the hybrid vigor and uniformity exhibited by F1 hybrids is lost in the next generation (F2). Consequently, seed produced by hybrids is consumed rather than planted.
- the rice dwarf mutant, d1 contains a non-functional RGA1 gene, encoding the GTP-binding ⁇ -subunit of the heterotrimeric G protein.
- This mutant was originally isolated as a spontaneous mutant with reduced height and shorter, erect, thicker, broad, dark green leaves, compact panicles, and short, round grains.
- the d1 plants present higher photosynthetic rates, stomatal conductance, and ⁇ leaf than wild type during both mild and severe water limitation, and have a greater number and percentage of tillers producing panicles, with resulting higher reproductive yield.
- the d1 plants also perform better than wild-type plants under high planting densities.
- FIG. 1 shows water stress during flowering and grain filling; mutant plants remain dark green and healthy while wild-type plants dry out and have a few living leaves under severe drought.
- the three watering conditions were: well-watered (100% soil relative water content), medium-water (45% soil relative water content), and low water (30% soil relative water content) conditions. Photographs were taken 140-160 days after emergence.
- FIG. 2 shows that d1 plants exhibit lower leaf temperatures than wild-type plants under all 3 watering conditions.
- Leaf temperatures on plants 165 days after emergence were measured by infrared thermography using a FLIR T620 Thermal Imaging Camera (FLIR Systems, USA). In order to obtain reliable comparisons, images were obtained over the shortest time interval possible; approximately 15 minutes total. Pseudo-colored temperature scales are indicated directly on the photographs.
- FIG. 3 shows that net photosynthesis and stomatal conductance are reduced more severely in wild-type plants than in d1 mutants under medium-water (45% soil relative water content) and low-water (30% soil relative water content) conditions, while these parameters are statistically identical under well-watered (100% soil relative water content) conditions. Parameters were measured on leaves between 120 and 130 days after emergence, using a Li-Cor 6400 Portable Photosynthesis System. Light intensity was 500 m ⁇ 2 s ⁇ 1 .
- FIG. 4 shows that d1 mutants have higher stomatal conductance but nevertheless lose less water (have lower transpiration rates) than wild-type plants. This is presumably due to the lower leaf temperatures of d1 plants as compared to wild-type (as shown in FIG. 3 ); lower leaf temperatures result in a reduced vapor pressure deficit, i.e. a reduced physical driving force for evaporative water loss. Measurements were made under ambient conditions of 500 ⁇ mol m ⁇ 2 s ⁇ 1 light and 30° C. temperature using a Li-Cor 6400 Portable Photosynthesis System. Measurements were taken on seedlings at 40 days after emergence. Each data point represents an individual plant. Lines depicted are regression lines for each genotype; ANCOVA analysis demonstrated significant differences (P ⁇ 0.05) in the slopes.
- FIG. 5 shows that the water potential ( ⁇ leaf ) of d1 plants is higher than that of wild-type plants under medium-water and low-water conditions, and equal to that of wild-type plants under well-watered conditions (the three watering regimes were specified above).
- ⁇ leaf measurements made using a Scholander pressure chamber, were taken from the flag leaf of the primary tiller of plants 120-130 days after emergence.
- FIGS. 3-5 show that d1 plants have improved water status and higher photosynthetic rates than wild-type plants under drought conditions.
- FIG. 6 is a graph showing the percentage of stalks flowering at the end of the reproductive phase in high water, low water and medium water conditions. One can see that water limitation decreases the number of stalks developing a flower head in wild-type plants but not in d1 mutants.
- FIG. 7 shows the average number of panicles produced through time in d1 and wild-type plants. Panicle production is greater in the d1 mutant than in wild-type plants. Water-limited conditions result in a pronounced reduction in the average number of panicles produced by wild-type plants but do not affect average panicle production in d1 plants.
- FIG. 8 shows drought-induced grain abortion per panicle in wild-type and d1 plants under the three watering regimes.
- the data are from 6 panicles randomly selected for analysis from 6 different plants of each genotype and water treatment combination, measured at the end of the life cycle.
- FIG. 9 shows that estimated grain yield per plant is statistically equal in wild-type and mutant d1 plants under well-watered conditions and is much higher in d1 mutants than in wild-type plants under drought conditions.
- FIG. 10 shows that in the absence of drought stress, increased planting density reduces photosynthesis at light saturation to a greater extent in wild-type than in the d1 mutant. Plants were grown in monospecific stands at the indicated planting densities. Net photosynthesis at 1500 ⁇ mol m ⁇ 2 s ⁇ 1 light was measured on the flag leaf of the primary tiller at 60 days after emergence using a Li-Cor 6400 Portable Photosynthesis System.
- FIG. 11 from the same experiment as FIG. 10 , shows that high planting density compromises plant growth and survival (as indicated by plant height) earlier in wild-type than in d1 plants.
- FIGS. 10 and 11 show that these traits are exhibited to a greater extent by the d1 mutant than by wild-type plants.
- Plant Heterotrimeric G Protein Function Insights from Arabidopsis and Rice Mutants Laetitia Perfus-Barbeoch, Alan M. Jones and Sarah M. Assmann
- Heterotrimeric G proteins have been implicated in a wide range of plant processes. These include responses to hormones, drought, and pathogens, and developmental events such as lateral root formation, hypocotyl elongation, hook opening, leaf expansion, and silique development. Results and concepts emerging from recent phenotypic analyses of G-protein component mutants in Arabidopsis and rice are adding to our understanding of G-protein mechanisms and functions in higher plants.
- G proteins provide a key mechanism by which a specific signaling cascade is switched on or off to translate an incoming signal into a specific cellular response.
- G proteins include the ⁇ , ⁇ , and ⁇ subunits of the G protein heterotrimer, possible heptahelical G-protein-coupled receptors (GPCRs), and regulator of G-protein signaling proteins (RGS).
- GPCRs possible heptahelical G-protein-coupled receptors
- RLS regulator of G-protein signaling proteins
- the G protein itself consists of three different subunits, ⁇ , ⁇ , and ⁇ (respectively named G ⁇ , G ⁇ , and G ⁇ ), which form a heterotrimeric complex in the inactive state. Binding of an agonist (i.e. an activating ligand) to its specific GPCR leads to the conversion of an inactive G protein to its active conformation.
- the GPCR acts as a guanine nucleotide exchange factor, causing G ⁇ to exchange GDP for GTP.
- G ⁇ -GTP separates from the G ⁇ dimer and both G ⁇ -GTP and the G ⁇ dimer separate from the receptor and can activate downstream effectors.
- GTPase-activating proteins GAPs
- G ⁇ is encoded by a single copy gene, designated GPA1 or RGA1, respectively [14,15].
- G ⁇ is likewise encoded by a single-copy gene, designated AGB1 or RGB1, respectively [16,17].
- AGG1 or RGG1 Two G ⁇ genes were recently isolated from Arabidopsis and rice: AGG1 or RGG1 [18,19] and AGG2 or RGG2 [19,20]. No plant gene has been found that is highly homologous to metazoan GPCRs.
- GCR1 is a likely candidate as a GPCR-encoding gene because it encodes a protein that has some GPCR sequence similarity and a predicted heptahelical structure that is the hallmark of bona fide GPCRs [8,21,22].
- RGS1 the Arabidopsis genome contains only one member of the RGS family, RGS1 [23].
- G ⁇ , G ⁇ , G ⁇ , GCR1 and RGS1 can all be found at the plasma membrane of plant cells [1,8,19,22,23,24,25].
- G ⁇ interacts tightly with both G ⁇ subunits in both Arabidopsis and rice [18,19,20].
- gel-filtration experiments have confirmed that G ⁇ dimers associate with G ⁇ . This association is disrupted by a non-hydrolysable form of GTP, GTP ⁇ S, which is expected to maintain the activated conformation of G ⁇ [19].
- Pandey and Assmann [8] used in planta and in vitro co-immunoprecipitation as well as split-ubiquitin yeast two-hybrid assays to provide the first conclusive evidence that the putative GPCR, GCR1, physically interacts with G ⁇ .
- RGS1 interacts with both a constitutively active GPA1 (GPA1 QL , the GTPase-deficient version of GPA1) and wild-type GPA1, and the carboxy-terminal domain of AtRGS1 has been shown to exert GAP activity on a yeast G ⁇ [23].
- Seed germination is a complex phenomenon that is modulated by numerous signals, including gibberellins (GA), abscisic acid (ABA), brassinosteroids (BR), ethylene, light, and sugars, some acting in concert and others in opposition [26].
- GA gibberellins
- ABA abscisic acid
- BR brassinosteroids
- gpa1-1 and gpa1-2 mutant seeds exhibit delayed germination [27], suggesting that they are more dormant than wild-type seeds. Consistent with this phenomenon, gpa1 mutants exhibit moderately increased sensitivity to the inhibition of germination by ABA and sugars [2,27]. Many of these phenotypes are also observed in Arabidopsis T-DNA insertional mutants of Pirin1, a cupin-domain protein that has been identified as a GPA1 interactor in yeast two-hybrid assays [27].
- gcr1 mutants Like gpa1 mutants, gcr1 mutants exhibit reduced sensitivity toward GA and BR in seed germination, whereas GCR1 overexpression reduces seed dormancy [22,29]. Under some but not all conditions, seeds of gcr1 gpa1 and gcr1 agb1 double mutants have additive or synergistic germination responses to GA and BR, which is unexpected if GCR1 were to function upstream of the G protein. Thus, under some conditions, GCR1 appears able to act independently of the heterotrimer in regulating seed germination [22].
- G ⁇ may also be a component of BR signaling in rice because BL-stimulated expression of a novel BL-enhanced gene is weaker in d1 mutants than in wild-type seedlings [33].
- Ueguchi-Tanaka et al. [32] suggest that there may be two separate GA-signaling pathways in rice, with either high or low sensitivity to GA, and that RGA1 may mediate the former pathway. Both this model and the ‘GPA1 modulation’ model described above [2,32] are consistent with the current data from both Arabidopsis and rice. Hence, additional experimentation, including determination of the ABA sensitivity of d1 seeds, will be required to distinguish between these two possibilities.
- Root growth and architecture involves a balance between cell production in the apical and lateral root meristems and the subsequent elongation of those cells.
- One advantage of the root as a model system for development is that it is possible to measure rates of cell production and elongation as well as the number of lateral root primordial quite precisely, thereby making it possible to quantify exactly what has changed in the roots of loss- and gain-of function G-protein mutants.
- the formation of lateral root meristems originates from a set of founder cells that differs from that used to form the primary meristem [34,35]. Therefore, it would not be surprising if the molecular mechanisms that underlie the initiation of lateral and primary root meristems were different. Studies on root meristem formation and maintenance using G-protein mutants are beginning to reveal these mechanistic differences.
- rgs1 mutants have longer primary roots because of their increased cell production rate in the primary root meristem [23 •• ].
- rgs1 cells which lack GAP activity, are predicted to have a greater steady-state pool of activated G ⁇ . This prediction is consistent with the observation that the expression of a transgene that encodes GPA1 QL also causes accelerated cell production by the primary root meristem [23 •• ]. This suggests that G ⁇ plays a role in modulating cell division in the primary root meristem.
- the lack of a large effect of the gpa1 null mutation on primary root growth suggests that the type of modulation that GPA1 exerts may be an increase over a basal state, a state that does not require Ga.
- primary root elongation In response to exogenous treatment with plant growth regulators such as ABA and auxin, primary root elongation is retarded and/or the direction of primary root growth changes.
- the primary root elongation of gpa1, agb1, or gcr1 single mutants, as well as of double and triple combinations of these mutants, is more sensitive to inhibition by ABA than that of wild-type plants ([8 •• ]; S Pandey, S M Assmann, unpublished).
- the auxin inhibition of primary root length in gpa1 and agb1 mutants is the same as that in wild-type plants [3 •• ], indicating that the dependency of growth inhibition on G proteins differs depending on the hormonal stimulus.
- auxin is a key activator of lateral root initiation [36].
- auxin agb1 plants form more lateral roots, whereas gpa1 plants form fewer lateral roots, compared to wild-type plants [3 •• ].
- auxin-induced phenotype for the proliferation of lateral roots is AGB1-dependent, ectopic expression of GPA1 (which is expected to sequester AGB1 into the heterotrimeric complex) also yields an agb1-like phenotype.
- the expression of GPA1 QL has no effect on this phenotype, a finding that is inconsistent with GPA1 acting as a positive modulator of cell division in the lateral root meristem.
- Ullah et al. [3 •• ] propose that free G ⁇ directly attenuates auxin-induced cell division in lateral roots, as opposed to G ⁇ acting to stimulate this process.
- gpa1 and agb1 seedlings When grown in darkness, gpa1 and agb1 seedlings have shorter hypocotyls than wild-type plants because of a reduction in cell number, and these seedlings exhibit partially opened hooks [1,3 •• ,37]. These phenotypes were also observed in gcr1 gpa1 double, agb1 gcr1 double, and agb1 gpa1 gcr1 triple mutants [22]. By contrast, rgs1 mutant seedlings have a longer etiolated hypocotyl as a result of increased cell elongation. This mutant phenotype is similar to that observed in plants that express GPA1 QL , consistent with the premise that, in plants as in animals, RGS proteins oppose G ⁇ activation [22].
- gpa1 and agb1 mutants When grown in light, gpa1 and agb1 mutants have rounded rosette leaves [1]. The round-leaf phenotype is also found in Arabidopsis gcr1 gpa1 and agb1 gcr1 double mutants and agb1 gcr1 gpa1 triple mutants [22]. Because gcr1 single mutants have wild-type phenotypes for both hypocotyl and rosette-leaf development, GCR1 may not act as the GPCR that is responsible for control of these developmental pathways [22].
- the rice G ⁇ (d1) mutants also exhibit an altered shoot morphology, consisting of broad, dark green leaves and compact panicles [5,30,31,32,38].
- Dwarf phenotypes are often associated with GA insensitivity, and GA induction of internode elongation is significantly reduced in the d1 mutants [31].
- the GA responsiveness of the elongation of the second leaf sheath is similar in d1 mutants and wild-type plants [32]. This selective impairment of GA signaling in d1 mutants suggests cell specificity in GA response, with some pathways being only marginally dependent on Ga.
- agb1-1 mutants have a floral phenotype consisting of shorter flowers and thicker siliques, but this phenotype is not shared by gpa1 mutants [1,3 •• ,39].
- Constitutive overexpression of GPA1 reduces silique length, producing a phenotype that is similar to that of agb1. This evidence is consistent with the idea that silique length is controlled by released Gbg [3 •• ].
- gpa1 sepals and pedicels are longer, whereas agb1 sepals are shorter, than those of wild-type plants [3 •• ], findings that are again consistent with a G ⁇ -dependent pathway.
- G proteins are implicated in several stress-signaling pathways in plants. In mature leaves, G proteins transmit signals to molecules, including small GTPases, ion channels, and phospholipases, that are effectors in the responses to various biotic and abiotic stress conditions, including pathogen elicitation, ozone treatment and water deficit.
- gpa1 null mutants and the double mutant gpa1-4 agb1-2 respond differently to O 3 compared to wild-type plants, and to gcr1 and rgs1 single mutants.
- the major difference observed among these mutant genotypes is an O 3 -resistant phenotype of the gpa1 lines, indicated by lack of leaf curling in response to O 3 [6].
- gpa1 mutants exhibit reduced O 3 sensitivity at the whole-leaf level.
- gpa1 mutants also exhibit aspects of ABA insensitivity, including reduced ABA inhibition of guard cell inward K + channels and altered ABA-promotion of slow anion currents [4].
- SIP sphingosine-1-phosphate
- gcr1 mutant guard cells exhibit hypersensitivity to ABA and SP in both inhibition of stomatal opening and promotion of stomatal closure [8 •• ], which would be unexpected if GCR1 were to transmit the ABA signal to GPA1.
- Pandey and Assmann [8 •• ] therefore proposed that GCR1 acts as a negative regulator of GPA1-mediated ABA responses in guard cells. Consistent with this phenomenon, gcr1 mutant plants have higher expression levels of some known drought- and ABA-regulated genes after exogenous ABA treatment and exhibit improved recovery following drought stress [8 •• ].
- PLCs phosphatidylinositol-phospholipase Cs
- PLDs phospholipase Ds
- PLD ⁇ 1 In Arabidopsis , PLD ⁇ 1 directly binds GPA1 via a motif similar to the DRY motif that is present in many mammalian GPCRs [50 •• ]. Binding inhibits PLD ⁇ 1 activity and is relieved upon GTP addition, suggesting that, in vivo, G protein activation leads to the activation of PLDa1 [50 •• ]. Thus, it will be of interest to assess PLC and PLD activity in guard cells in which the levels of G-protein components are altered.
- the different phenotypes of gpa1 and agb1 mutants could reflect differences in the relative levels of the released subunits from the heterotrimeric complex, different fluxes of signaling through G ⁇ (as opposed to G ⁇ ) in the different cell types or organs, and/or a different relative balance in positive or negative feedback.
- G ⁇ the relative levels of the released subunits from the heterotrimeric complex
- G ⁇ the relative levels of the released subunits from the heterotrimeric complex
- G ⁇ as opposed to G ⁇
- a different relative balance in positive or negative feedback To sort out these issues, it will be necessary to determine the effect on a given trait of quantitatively altered ratios of G ⁇ to G ⁇ , rather than of the two extremes of ratios of 0 or ⁇ that are created by single null mutations.
- GCR1 is the sole candidate GPCR to be identified in Arabidopsis on the basis of homology criteria and its sequence similarity to known GPCRs is limited.
- G proteins may be transduced either via novel GPCRs or through proteins that transmit signals to G proteins independently of GPCRs [51,52].
- plant-specific ‘unconventional’ G proteins such as A. thaliana Extra Large G Protein1 (XLG1), a protein that has significant similarity to G ⁇ subunits and exhibits GTP-binding capability [11,53]
- XLG1 A. thaliana Extra Large G Protein1
- the future is bright for model plant systems such as Arabidopsis and rice to contribute new insights regarding this ubiquitous eukaryotic signaling paradigm.
- GCR1 may positively regulate seed germination by coupling BR promotion of GA-stimulated germination. GCR1 also can act independently of GPA1 and AGB1 in a pathway to regulate GA-stimulated germination [23 •• ]. RGS1 antagonizes the activation of GPA1 [23 •• ]. Pirin1 may positively regulate seed germination by overcoming the negative effect of ABA or by activating germination-promoting pathways [27′]. In rice, RGA1 may work in a high-sensitivity GA pathway that regulates the induction of Ca 2+ -ATPase and ⁇ -amylase, leading to seed germination [32]. RGA1 may also be a component of BR signaling [33].
- GPA1, AGB1 and GCR1 may act in the inhibition of primary root development by ABA ([8 •• ]; S Pandey, S M Assmann, unpublished). Furthermore, GCR1 negatively regulates ABA-induced gene expression [8 •• ]. AGB1 and GPA1 activate cell division in both hypocotyls and leaves [1,2,3 •• ,37] whereas RGS1 antagonizes the activation of GPA1 in apical root meristems [23 •• ].
- Auxin treatment also increases GPA1 transcript levels and decreases AGB1 transcript levels (not shown in figure, [3 •• ]).
- AGB1 functions downstream of GPA1 and inhibits auxin-induced cell division, and GPA1 inhibits AGB1 function [3 •• ].
- ABA triggers SP formation, which is coupled by GPA1 to inhibit plasma membrane inwardly rectifying K+ channels and to activate slow anion channels, resulting in the inhibition of stomatal opening and the promotion of stomatal closure [4,45 •• ].
- the GPCR-like protein GCR1 directly binds to GPA1 and negatively controls ABA- and S1P-regulation of stomatal apertures [8].
- responses to avirulent rice blast fungus including the accumulation of transcripts for the small GTPase, OsRac1, are attenuated in the RGA1 mutant d1.
- OsRac1 acts as a key molecular switch for multiple signaling pathways, such as the production of reactive oxygen species that lead to disease resistance.
- Phenotypes of Daiokoku dwarf1 (d1) mutants (DK22, Mutant loss of function for G ⁇ subunit in rice ( Oryza sativa ) HO541, CM 1361-1, T65d1, rga1) DK 22 Nipponbare Point mutation of G598 to T in 8 th Stop codon generated Shorter and rounded grains [30, 31]. exon. [31, 40]. Reduced GA and BL strategymlation of gene expression [32, 33]. Protein null [19*]. Ho 541 Nipponbare Spontaneous mutant: Deletion of RGA1 transcript null [3-] Shorter and darker green leaves, more compact particle [5, 30-32, 833 basepairs between 1 st exon and 38]. intron.
- CM 1361-1 Kinmaze Insertion between nucleotides 354-355 Predicted protein lacks Shorter internodes - may be due to a decrease in the number GTP-, effector- and of cells per internode [31].
- receptor-binding regions Reduced GA stimulation of internode growth [32]. [31]. Normal GA stimulation of second leaf sheath elongation [32]. T65d1 Taichung 65 Deletion of nucleotides 1003-1004. Stop condon generated Reduced hypersensitive response to infection by rice blast before third effector-binding fungus [5]. region [32]. rga1 Nipponbare Antisense suppression.
- RGA1 transcript null line Increased sensitivity to infection by virulent strain of [31] bacterial blight [7].
- HO 532 HO 533 HO 537 Nipponbare Spontaneous mutants.
- HO 538 HO 552 FL2 Nipponbare Marker line derived from HO 538.
- ID 1 Shiokari Deletion of nucleotides 1003-1004.
- Not used for phenotypic analysis CM392; Kinmaze Induced by N-methyl- [5, 30] Not used for phenotypic analysis. 1729; 248; N-nitrosourea. 723; 1232 DKT 1 Taichung 65 Point mutation of A1075 to T.
- GPA1 ⁇ Ws 35S promoter :constitutive form of Overexpression of Increased hypocotyl length caused by increased cell GPA1 cDNA constitutively active G ⁇ elongation [23**].
- GPA1 QL Longer primary roots caused by increased cell production [23**].
- cG ⁇ Ws DEX inducible Overexpression of Under low light condition shorter hypocotyls are caused by a promoter::constitutive form of constitutively active G ⁇ reduction of cell elongation, also smaller cotyledons and GPA1 cDNA.
- GPA1 QL [54]. increased stomatal density in hypocotyl [54].
- wG ⁇ Ws DEX inducible promoter Overexpression of full- Under low light condition, shorter hypocotyls are caused by a GPA1 cDNA. length GPA1 protein [54]. reduction of cell elongation, also smaller cotyledons and increased stomatal density in hypocotyl [54].
- gpa1 Col-0 DEX inducible promoter Complementation in gpa1 Hypersensitive to GA stimulation of germination [2].
- GPA1 GPA1 cDNA background [3**, 37]. In darkness, shorter hypocotyls [37]. Hypocotyl hypersensitive to auxin-induced adventitious root formation [3**].
- GOX Col DEX inducible promoter Overexpression of GPA1 Mimics agb1-2 lateral root phenotype: more lateral roots GPA1 cDNA: [2, 37]. [3**].
- GOX1 Tobacco cells DEX inducible promoter :: Transformed BY2 cells Shorter cell cycle [1]. Nicotiana GPA1 cDNA. overexpressing GPA1 Higher Ptdlns-PLC activity [49]. tabaccum cv. [1, 49]. Higher Ins(1, 4, 5)P 3 content [49].
- Missense mutation larger because of splicing More sensitive to the GA biosynthesis inhibitor paclobutrazol failure to splice out the 1 st intron. failure. Stop condon [2, 22]. Hypersensitive to sugar inhibition of germination [2]. generated [39]. In darkness, partial de-etiolation: open hook; shorter hypocotyls caused by reduced cell division [1]. Hypocotyl hypersensitive to auxin-induced adventitious root formation [3**]. Primary root forms more lateral root primordia [3**]. agb1-2 Col-0 T-DNA insertion in 4 th exon. Lacks full-length transcript More sensitive to auxin promotoino of lateral root formation Salk collection. [3**]. [3**].
- BOX Col-0 DEX inducible promoter Overexpression of AGB1 AGB1 cDNA. [3**, 37]. Mutant loss of function for GCR 1 in Arabidopsis Phenotypes of gcrf mutants gcr1-1 Col-0 T-DNA insertion in 8 th intron. Lacks full-length transcript Less sensitive to GA and BR stimulation of germination [22]. Salk collection. [22]. Hypersensitive to ABA inhibition of germination [8**]. gcr1-2 Col-0 T-DNA insertion in 6 th exon. Lacks full-length transcript Hypersensitive to the GA inhibitor paclobutrazol [22]. Salk collection. [22]. Increased ABA promotion of ABA-regulated gene expression [8**].
- gcr1-3 Ws T-DNA insertion in 2 nd intron. Lacks full-length Increased resistance to drought stress [8**].
- gcr1-4 Col T-DNA insertion in 3 rd intron. Lacks full-length transcript Hypersensitive to ABA and S1P promotion of stomatal SAIL collection of TMRI. [8**]. closure [8**]. Flowers slightly earlier [22].
- GCR1 Mutant gain of function for GCR1 in Arabidopsis Phenotypes of GCR1 overexpressors gcr1-3 Ws DEX inducible promoter::GCR1 Expression in gcr1-3 GCR1-FLAG immunoprecipitates with GPA1 [8**].
- GCR1 cDNA fused with FLAG tag. background [8**].
- GCR1-over- Col 35S promoter::GCR1 Overexpression of GCR1 Lacks seed domancy [29]. expressing cDNA [29]. Increawsed expression of germination associated genes [29].
- ROX Col-0 DEX inducible promoter Overexpression of full Mimics gpa1 mutant hypocotyl phenotype under darkness: RGS1 open reading frame length RGS1 protein [23**]. shorter hypocotyl [23**]. Hypersensitive to 6% D-glucose inhibition of seedling growth [23**]. Loss of function double/triple mutants in Arabidopsis Phenotypes double/triple mutants gpa1-4 Col-0 Cross between gpa1-4 and agb1-2. [37] Less sensitive to GA and BR stimulation of germination agb1-2 (same sensitivity as agb1 mutant [22]). Shorter hypocotyls and partially opened hooks [37].
- agb1 leaf morphology rounded lamina shape [22].
- gpa1 and agb1 phenotype under darkness shorter hypocotyl and partially opened hooks [22].
- gpa1 and agb1 leaf morphology rounded lamina shape [22].
- agb1-1 Col Cross between agb1-1 and [39] Shorter petiole, shorter lamina than either agb1 or er105 er-105 receptor-like kinase erecta mutant, single mutant, suggesting that ER and AGB1 function in er-105 parallel pathways controlling these characteristics [39].
- 35S cauliflower mosaic virus 35S promoter
- BOX AGB1 overexpressing lines
- Col Columbia
- DEX dexamethansone
- elk4 erecta like 4 mutant
- er-105 receptor-like kinase erecta mutant
- GFP green fluorescent protein
- GOX GPA1 overexpressing lines
- Ins(1,4,5)P 3 inositol-1,4,5-trisphosphate
- PtdIns-PLC phosphatidylinositol-phospholipase C
- ROX RGS1 overexpressing lines
- SAIL Syngenta Arabidopsis Insertion Library
- slr slender rice mutant
- TMRI Torrey Mesa Research Institute
- Ws Wassilewskija.
- Phenotypic plasticity is the ability of one genotype to display different phenotypes under different environmental conditions. Although variation for phenotypic plasticity has been document in numerous species, little is known about the genetic mechanisms underlying phenotypic plasticity. Given their widespread roles in hormonal and environmental signaling, we examined whether genes which encode heterotrimeric G proteins are plasticity genes.
- G-protein mutations did not affect significantly the amount of phenotypic variation within an environment for any trait, but did affect significantly the amount of phenotypic plasticity for certain traits.
- AGB1 which encodes the 13 subunit of the heterotrimeric G protein in Arabidopsis , is a plasticity gene and regulates reproductive trait plasticity in response to water availability, resulting in increased fitness (defined as seed production) under drought stress.
- GTP multisubunit guanosine triphosphate
- GPCR membrane-bound G-protein-coupled receptor
- the Arabidopsis thaliana genome contains single genes encoding G ⁇ (GPA1) (Ma et al., 1990) and Gb (AGB1) (Weiss et al., 1994) subunits, and two known G ⁇ (AGG1 and AGG2) (Mason & Botella, 2000, 2001) genes.
- GPCR diversity is also reduced in Arabidopsis ; only one putative GPCR, GCR1, has been functionally characterized (Pandey & Assmann, 2004), although several dozen additional genes have been predicted to function as GPCRs based on topology prediction (Moriyama et al., 2006; Gookin et al., 2008) and coupling to GPA1 in yeast (Gookin et al., 2008).
- Such variants may be mechanistically related to plant-specific aspects of the G-protein complement, including the slow GTPase activity of the plant G ⁇ subunit (Johnston et al., 2007; Pandey et al., 2009), the existence of an unusual class of plant-specific G ⁇ -like proteins, the extra-large G proteins (Lee & Assmann, 1999; Pandey et al., 2008) and the presence in Arabidopsis of novel GPCR-like regulatory proteins, the GTG and RGS proteins (Chen et al., 2003; Pandey et al., 2009).
- G-protein signaling Assmann, 2004. Further characterization of GTGs and other unconventional G proteins, such as the XLGs (Lee & Assmann, 1999; Ding et al., 2008), and the identification of GPCR ligands, potential tissue-specific GPCRs, and G-protein signaling effectors in plants may help to elucidate this question.
- G proteins may serve as signal modulators instead of direct transducers of signals ( FIG. 1 ).
- G proteins could fine tune a phenotype or physiological response based on multiple signals/environmental inputs (Assmann, 2004).
- This model also addresses another paradox in plant G-protein signaling: why, if G proteins are important to plant physiology, are G-protein mutations not lethal in plants? A mutation may not be lethal if additional copies or similar versions of the gene exist in the genome or if the expression of the gene is specific to a certain stress (e.g. cold shock) or to a less vital plant tissue or cell type (e.g. trichomes).
- a certain stress e.g. cold shock
- a less vital plant tissue or cell type e.g. trichomes
- G ⁇ and G ⁇ are both encoded by single, canonical genes which are widely expressed throughout the plant (Ma et al., 1990; Huang et al., 1994; Lease et al., 2001; Anderson & Botella, 2007).
- G proteins function in directing hormonal and environmental cross-talk, they may be required only in the production of the ‘optimal’ phenotype, and the direct transducers of the signal would still function in the absence of functional G-protein subunits.
- Plants, being sessile organisms, are hypothesized to have evolved increased phenotypic plasticity, the ability of one genotype to display different phenotypes under different environmental conditions, compared with their mobile animal counterparts (Bradshaw, 1972; Schlichting, 1986; Sultan, 1987; Huey et al., 2002). Heightened plasticity in plants would allow plants to compensate for inescapable and inhospitable environments. Although variation for plasticity has been documented in numerous plant and animal species, and many theories have been proposed concerning the ecological and evolutionary significance of this variation, very little is known about the explicit genetic machinery which underlies phenotypic plasticity.
- the phenotypic plasticity (or lack thereof) of a trait can be graphically represented by a reaction norm, which is a plot of the mean phenotypic value of the trait in different environmental conditions ( FIG. 2 ).
- a horizontal reaction norm indicates that the trait lacks plasticity, whereas a line with a nonzero slope or a curved line is indicative of phenotypic plasticity.
- plasticity genes control the shape or slope of the reaction norm of a trait, when these genes are mutated, it is expected that the shape/slope of the reaction norm will diverge from that of the wild-type.
- Mutations may alter the height of the reaction norm without affecting the overall plasticity for a trait (line C), affect the amount of plasticity for a trait (line B) or may change the direction of plasticity for a trait (line D).
- Differences in reaction norm shapes (plasticities) among genotypes can be detected and tested using analysis of variance (ANOVA)-based statistical methods. Specifically, a significant genotype ⁇ environment/treatment interaction term for a trait indicates that there is variation for plasticity among the genotypes, that is, the response curves have different shapes/slopes.
- HY1 and HY2 which encode a plastid heme oxygenase and a phytochromobolin synthase, respectively
- HY1 and HY2 which encode a plastid heme oxygenase and a phytochromobolin synthase, respectively
- HY1 and HY2 are plasticity genes.
- naturally occurring polymorphism of the photochrome PHYB locus has been associated with altered light responses in Arabidopsis ; however, phenotypic plasticity was not measured explicitly (Filiault et al., 2008).
- Plasticity has also been documented among wild-type accessions of Arabidopsis (Pigliucci & Kolodynska, 2002; Schmuths et al., 2006; Brock & Weinig, 2007), and naturally occurring variation for plasticity within plant species has been assessed in quantitative trait locus (QTL)-based phenotypic plasticity studies. Using recombinant inbred lines in lieu of genetic mutants, these studies have lent additional support for the existence of plasticity genes.
- QTL quantitative trait locus
- the signaling cross-talk mechanism discussed above may be an important component of phenotypic plasticity, as phenotypes could be tweaked on the basis of multiple environmental inputs.
- a mutation in a G-protein subunit might decrease cross-talk and therefore reduce the plant's ability to adjust a phenotype or response to changing environments based on multiple signals.
- a G-protein mutation might affect the degree of plasticity of a trait ( FIG. 2 , line B), the amount of variation within an environment for a trait (that is the ‘noisiness’ of the trait) or might simply shift the mean value of the trait away from the wild-type value ( FIG. 2 , line C) (Assmann, 2004).
- G-protein genes are plasticity genes, as well as evaluate the importance of G proteins to plant fitness.
- previous studies of heterotrimeric G proteins have, to a large extent, focused on guard cell physiology (Wang et al., 2001; Pandey & Assmann, 2004; Fan et al., 2008) and cell division (Ullah et al., 2001, 2003; Chen et al., 2006).
- guard cell physiology Wang et al., 2001; Pandey & Assmann, 2004; Fan et al., 2008
- cell division Ullah et al., 2001, 2003; Chen et al., 2006.
- gpa1-3 and gpa1-4 are null mutants at both the transcript and protein levels (Jones et al., 2003).
- agb1-2, gcr1-1 and gcr1-2 are all transcript null alleles (Ullah et al., 2003; Chen et al., 2004).
- agb1-1 produces a larger and less abundant AGB1 transcript compared with the wild-type as a result of a destabilizing point mutation, which results in a failure to splice out the first intron and the introduction of a premature stop codon (Lease et al., 2001). All alleles were backcrossed once in our laboratory and the genotypes of parent plants were confirmed via PCR of genomic DNA.
- Seed storage was identical for all seed lots. Cold stratified seeds (stratified at 4° C. for 48 h in darkness on wet filter paper) were sown directly on the surface of a soil mix composed of Miracle-Gro potting mix (The Scotts Co, Marysville, Ohio, USA), Turface Greens Grade fritted clay (Profile Products LLC, Buffalo Grove, Ill., USA) and perlite in a 16:8:1 volume ratio. The plants were grown in Kord 90 mm press-fit pots (Kord Products Inc. Brampton, Ontario, Canada) in a walk-in Conviron growth chamber (Conviron Inc. Winnipeg, Manitoba, Canada). The photoperiod was 12 h light (140 ⁇ mol m ⁇ 2 s ⁇ 1 , 21° C.) and 12 h dark (19° C.) and the relative humidity was 60%.
- Miracle-Gro potting mix The Scotts Co, Marysville, Ohio, USA
- Turface Greens Grade fritted clay (Profile Products LLC, Buffalo Grove, Ill.
- Plants were arranged in a split-plot design. Three trays, each representing one water level, were clustered in a block, and 12 blocks were placed on separate shelves in the growth chamber. Genotypes were randomly assigned a position within a tray with two genotype replicates per tray. There were two replicate plants ⁇ 8 genotypes ⁇ 3 treatments ⁇ 12 blocks for a total population of 576 plants. The transition from vegetative to reproductive growth was assessed by recording the flowering time (days from sowing), when the first open flower was visible, for each plant. Because of the large population size and the fact that flowering times were affected by treatment and genotype, each plant was individually harvested 4 wks.
- inflorescence height cm
- number of primary lateral branches and number of fruits plus any pistil which showed elongation or swelling.
- Excised rosettes were dried at 70° C. until a constant mass was achieved, and the dry mass was determined.
- seed number per fruit was determined using a dissecting scope. Aborted or shriveled seeds were excluded from the seeds per fruit measurements. Seed production was estimated for each plant in the three blocks which had seeds per fruit measurements as (total fruit number ⁇ seeds per fruit). Relative fitness (total seed number ⁇ mean total seed number for water level) was calculated post hoc for the three blocks for which total seed production was determined.
- Multivariate and univariate analyses were performed using Proc GLM in SAS 9.1 (SAS Inc. Cary, N.C., USA). As plant mortality resulted in an unbalanced design, the two genotype ⁇ treatment replicates within a block were averaged to enable analysis by Proc GLM. Multivariate analysis of variance (MANOVA) was first performed to identify whether there were significant effects of genotype, treatment, and genotype ⁇ treatment interaction for a suite of reproduction-related traits, including rosette mass, inflorescence height, lateral branch number and fruit number. A second MANOVA was performed using data only from the three blocks for which the seed number per fruit and total seed production were calculated.
- MANOVA Multivariate analysis of variance
- the second MANOVA included the following response variables: rosette mass, inflorescence height, lateral branch number, fruit number, seed number per fruit and total seed production.
- Univariate ANOVAs were performed following the MANOVAs in order to determine which traits showed significant variation for phenotypic plasticity (genotype ⁇ treatment interaction). Flowering time was analyzed using ANOVA only.
- the split-plot experimental design required that the whole-plot factor, treatment, be tested over the whole-plot random error term, treatment ⁇ block. Genotype and genotype ⁇ treatment interaction were tested over the residual error. Data and residuals were examined to ensure that all ANOVA assumptions were satisfied. Fruit number and total seed production required transformation (square root) in order to satisfy ANOVA assumptions of normality and stable variance.
- the plasticities of the gpa1-4 agb1-2 double mutant for the most part resembled those of the single agb1 mutants.
- gpa1 mutants showed increased plasticity for inflorescence height relative to Col ( FIG. 3 ).
- the double mutant showed reduced plasticity relative to gpa1 for inflorescence height ( FIG. 3 ), indicating that the double mutant shows an intermediate phenotype.
- gcr1 mutants showed increased plasticity for square root fruit number relative to the wild-type ( FIG. 4 ).
- AGB1 Functions as a Plasticity Gene for a Number of Reproduction-Related Traits
- AGB1 has been shown previously to function in inflorescence and fruit development.
- agb1-1 was originally isolated in a screen for erecta-like mutations, where it showed a slightly reduced inflorescence height and significantly shortened, blunt-tipped fruits relative to the wild-type, phenotypes which were later also observed in agb1-2 (Lease et al., 2001; Ullah et al., 2003).
- AGB1 was expressed ubiquitously throughout the plant, but its expression was elevated in flowers and highest in fruits (Lease et al., 2001).
- the shortened fruit phenotype corresponds to our findings that agb1 shows reduced seeds per fruit.
- This short phenotype is specific to agb1 mutants and is not observed in gpa1, agg1 and agg2 mutants (Ullah et al., 2003; Trusov et al., 2008). Functional selectivity of the G ⁇ subunit has been reported for other G-protein-mediated responses, including necrotrophic pathogen resistance (Llorente et al., 2005; Trusov et al., 2006), sugar inhibition of seed germination and lateral root formation (Chen et al., 2006).
- G-protein signaling activation of GPA1 results in a conformational change in G ⁇ and the release of the G ⁇ dimer, and signal propagation.
- G ⁇ is responsible for signal integration or transduction, resulting in wild-type plasticity for the number of fruits, seed number per fruit and total seed production.
- the a subunit typically requires the ⁇ subunit, not only for trimer reassembly, but also for GPCR association.
- GPA1 activation is eliminated by both gpa1 and agb1 mutations, but AGB1 activation is abolished only by the agb1 mutation.
- gpa1 and agb1 display opposite phenotypes (enhanced and reduced plasticity, respectively), which also suggests that AGB1 is responsible for mediating wild-type plasticity, as free AGB1 is active in gpa1 mutants (potentially more so than in the wild-type), but is absent from agb1 mutants.
- gcr1 mutants showed increased plasticity relative to the wild-type for fruit number and, although GCR1 might function in the mediation of plasticity for fruit number, there are probably additional unknown GPCRs that contribute to the perception and/or integration of environmental input signals with regard to the regulation of phenotypic plasticity.
- a G-protein mutation might increase or decrease the ability of a cell to buffer itself against stochasticity, and therefore it has been hypothesized that G-protein mutations might affect the amount of phenotypic variation within an environment (Assmann, 2004).
- phenotypic variances Table S2
- Table S3 Supporting Information
- gpa1 and agb1 are hyposensitive with regard to the ABA inhibition of stomatal opening, but show wild-type ABA promotion of stomatal closure (Wang et al., 2001; Fan et al., 2008), and gcr1 mutants are hypersensitive towards both ABA inhibition of stomatal opening and promotion of closure (Pandey & Assmann, 2004), our detailed phenotypic analysis of whole-plant traits under controlled water stress conditions did not support the predictions that would be made based on these stomatal response phenotypes.
- gcr1 mutants despite their stomatal hypersensitivity to ABA and their reported improved recovery following drought stress (Pandey & Assmann, 2004), showed no fitness advantage relative to the wild-type under drought stress or well-watered conditions. Although we predicted that gpa1 and agb1 mutants would show reduced fitness under drought stress based on the partial ABA insensitivity of their stomatal phenotypes, agb1 mutants (but not gpa1 mutants) exhibited increased fitness under drought stress and reduced fitness under well-watered conditions. The pleiotropic nature of G-protein mutations may, in part, explain why the stomatal response phenotypes were not predictive of plant fitness under drought stress.
- G proteins regulate stomatal density GPA1 as a positive modulator and AGB1 as a negative regulator (Zhang et al., 2008a), which may have implications for water loss and carbon assimilation under different environmental conditions.
- agb1 mutants and, to a lesser degree, gpa1 mutants are hypersensitive to ABA inhibition of germination and root growth (Pandey et al., 2006), which may contribute to survival under drought stress.
- agb1 mutants and, to a lesser degree, gpa1 mutants are hypersensitive to ABA inhibition of germination and root growth (Pandey et al., 2006), which may contribute to survival under drought stress.
- the lack of plasticity of agb1 in reproductive-related traits resulted in a fitness advantage under drought stress, lack of plasticity was maladaptive for agb1 under well-watered conditions.
- G proteins do contribute to plant fitness, in part via the regulation of phenotypic plasticity. Given that climate change can lead to increased variability in environments and resources, this finding could have important agronomical implications, as agb1 mutants show reduced plasticity and therefore more stable yields across environments.
- G proteins mediate responses to ABA, auxin, brassinosteroids, gibberellins and sugars, environmental signals, including ozone and pathogens, and intrinsic, unknown developmental cues mediating leaf shape, stomatal density and fruit shape.
- a fundamental question in G-protein signaling is how only two heterotrimeric G-protein combinations (GPA1/AGB1/AGG1 and GPA1/AGB1/AGG2) can transduce such a diversity of signals.
- G proteins function, at least in part, as cross-talk hubs, integrating signals, rather than directly transducing them (Assmann, 2004), thereby tweaking a phenotype relative to the environment at hand.
- RGA1 coding region sequence SEQ ID NO: 1 ATGGGCTCATCCTGTAGCAGATCTCATTCTTTAAGTGAGGCTGAA ACAACCAAAAATGCAAAATCTGCAGACATTGACAGGCGAATTTTG CAAGAGACAAAAGCAGAGCAACACATCCACAAGCTCTTACTTCTT GGTGCGGGAATCAGGGAAGTCTACGATATTTAAACAGATTAAG CTCCTTTTCCAAACTGGCTTTGATGAGGCAGAACTTAGGAGCTAC ACATCAGTTATCCATGCAAACGTCTATCAGACAATTAAAATACTA TATGAAGGAGCAAAAAAGAACTCTCACAAGTGGAATCAGATTCCTCA AAGTATGTTATATCCCCAGATAACCAGGAAATTGGAGAAAAACTA TCAGATATTGGAGATAACCAGGAAATTGGAGAAAAACTA TCAGATATTGGAGATAACCAGGAAATTGGAGAAAAACTA TCAGATATTGGAGATAACCAGGAAATTGGAGAAAAACTA TCAGATATTGAAA
- RGA1 protein sequence SEQ ID NO: 3 MGSSCSRSHSLSEAETTKNAKSADIDRRILQETKAEQHIHKLLLL GAGESGKSTIFKQIKLLFQTGFDEAELRSYTSVIHANVYQTIKIL YEGAKELSQVESDSSKYVISPDNQEIGEKLSDIDGRLDYPLLNKE LVLDVKRLWQDPAIQETYLRGSILQLPDCAQYFMENLDRLAEAGY VPTKEDVLYARVRTNGVVQIQFSPVGENKRGGEVYRLYDVGGQRN ERRKWIHLFEGVNAVIFCAAISEYDQMLFEDETKNRMMETKELFD WVLKQRCFEKTSFILFLNKFDIFEKKIQKVPLSVCEWFKDYQPIA PGKQEVEHAYEFVKKKFEELYFQSSKPDRVDRVFKIYRTTALDQK LVKKTFKLIDESMRRSREGT- d1 predicted protein sequence (based on our c
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Abstract
The invention provides for compositions and methods for producing plants that have higher yield in drought conditions by manipulating the G-proteins such as the rice alpha subunit gene, RGA1. In particular, the present invention provides for plants, varieties, lines, and hybrids, as well as plant tissues and plant seeds that contain modified G-protein activity, particularly RGA1 activity to engineer drought tolerance and improved seed production in plants, as well as improved tolerance to high density planting
Description
- This application is a Divisional application of U.S. Ser. No. 13/866,401 filed Apr. 19, 2013, which claims priority under 35 U.S.C. §119 to provisional application Ser. No. 61/665,385 filed Jun. 28, 2012, herein incorporated by reference in its entirety.
- Rice is one of the three major cereal crops and is a staple food for more than a third of the world's population. Drought is one of the major abiotic stress factors limiting crop productivity worldwide. Global climate changes may further exacerbate the drought situation in major crop-producing countries. Although irrigation may in theory solve the drought problem, it is usually not a viable option because of the cost associated with building and maintaining an effective irrigation system, as well as other issues, such as the general availability of water. Thus, alternative means for alleviating plant water stress are needed.
- Upon exposure of plants to drought conditions, many stress-related genes are induced and their products are thought to function as cellular protectants of stress-induced damage. The expression of stress-related genes is largely regulated by specific transcription factors. Members of the AP2, bZIP, zinc finger, and MYB families have been shown to have regulatory roles in stress responses. The rice and Arabidopsis genomes code for more than 1300 transcriptional regulators, accounting for about 6% of the estimated total number of genes in both cases. About 45% of these transcription factors were reported to be from plant-specific families (Riechmann et al., (2000) Science 290: 2105-2110).
- Plant modification for enhanced drought tolerance is mostly based on the manipulation of either transcription and/or signaling factors or genes that directly protect plant cells against water deficit. Despite much progress in the field, understanding the basic biochemical and molecular mechanisms for drought stress perception, transduction, response and tolerance remains a major challenge. Utilization of the knowledge on drought tolerance to generate plants that can endure under extreme water deficit condition is even a bigger challenge.
- Rice is a staple food for greater than one third of the world's population and worldwide total rice planting area is approximately 1.5 million square kilometers. In 2009 global rice production was over 670 million tons, second only to maize.
- Approximately 20% of rice growing areas worldwide are prone to drought. Drought is a particularly important issue for rice production. About 5000 liters of water are needed to produce one kilogram of rice, approximately double the needs of other crops.
- Despite efforts to develop drought-tolerant rice plants, very few attempts have been shown to improve grain yields. Examples of positive effects include transgenic rice plants expressing SNAC1 (Hu et al., (2006) Proc Natl Acad Sci USA 103: 12987-12992) and OsLEA3 (Xiao at al., (2007) Theor Appl Genet 115: 35-46), which was shown to improve grain yield under field drought conditions.
- Heterotrimeric G-proteins are key signal transduction components that couple the perception of an external signal by a G-protein coupled receptor (GPCR) to downstream effectors. More than one third of mammalian signaling pathways depend on heterotrimeric G-proteins, including vision, taste, olfaction, hormones, and neurotransmitters. G-protein coupled signaling pathways are targets of approximately half of all pharmaceuticals.
- The G-protein complex is comprised of Gα, Gβ and Gγ monomeric subunits that assemble as a heterotrimer that physically associates with a GPCR. Activation of the GPCR triggers the Gα subunit to exchange GDP for GTP, thus activating the G-protein. Once active the heterotrimeric complex dissociates from the GPCR and the Gα subunit separates from the Gβγ heterodimer. Both GTP-bound Gα and the Gβγ heterodimer transduce the signal to downstream effectors.
- Heterotrimeric G-proteins have been studied extensively in animals. To date, 23 Gα, 6 Gβ and 11 Gγ genes have been reported in mammals (Vanderbeld and Kelly (2000) Biochem. Cell Biol. 78: 537-550). The alpha subunits are classified into four subfamilies: Gs, Gi, Gq, and G12. In contrast, relatively little is known about the role G-proteins play in plants. Loss-of-function mutants in the Gα subunit of rice and Arabidopsis are completely viable, but show several characteristic developmental attributes. The rice mutant exhibits shortened internodes, rounded seeds, and partial insensitivity to gibberellin, whereas the Arabidopsis mutants have rounded leaves and altered sensitivity to a number of phytohormones (Ashikari et al. (1999) Proc. Natl. Acad. Sci. 96: 10284-10289; Fujisawa et al. (1999) Proc. Natl. Acad. Sci. 96:7575-7580; Ueguchi-Tanaka et al. (2000) Proc. Natl. Acad. Sci. 97: 11638-11643; Wang et al. (2001) Science 292: 2070-2072; (Ullah et al. (2001) Science 292: 2066-2069). A loss-of-function mutant in the Gβ subunit of Arabidopsis (AGB1) exhibits several defects including short, blunt fruits, rounded leaves, and shortened floral buds (Lease et al. (2001) Plant Cell 13: 2631-2641).
- It can be seen that there is a continuing need to develop drought tolerance in plants, particularly rice.
- It is an object of the present invention to modulate Gα proteins such as RGA1, in plants to engineer drought tolerance and increase seed yield under such conditions.
- Other objects will become apparent from the description of the invention, which follows.
- The rice dwarf mutant, d1, contains a non-functional RGA1 gene, encoding the GTP-binding α-subunit of the heterotrimeric G protein. Rice RGA1 encodes a 380 amino acid Gα protein. Applicants have identified a 2 bp deletion (allele d1), which results in a frameshift mutation resulting in a predicted protein truncation after amino acid 304. This mutant was originally isolated as a spontaneous mutant with reduced height and shorter, erect, thicker, broad, dark green leaves, compact panicles, and short, round grains. Applicants have identified that a non-functional RGA1 gene or other related G-proteins can be used to create higher seed production and yield under drought conditions. The d1 plants also perform better at higher planting density.
- Thus the present invention provides for compositions and methods for producing plants that have higher yield in drought conditions by manipulating the G-proteins such as the RGA1 gene. In particular, the present invention provides for plants, varieties, lines, and hybrids, as well as plant tissues and plant seeds that contain modified G-protein activity, particularly RGA1 activity to engineer drought tolerance and improved seed production in plants.
- In one embodiment, the present invention provides for one or more plants whose germplasm has been modified to render a G-protein, particularly the RGA1 gene or its gene product inactive. Moreover, in further embodiments the invention relates to the offspring (e.g., F1, F2, F3, etc.) of a cross of said plant wherein the germplasm of said offspring has the same mutation as the parent plant. Therefore, embodiments of the present invention provide for plant varieties/hybrids whose germplasm contains a mutation, such that the phenotype of the plants is non-functional RGA1 gene activity in drought conditions. In some embodiments, said offspring (e.g., F1, F2, F3, etc.) are the result of a cross between elite lines, at least one of which contains a germplasm comprising a mutation that renders the G-protein such as RGA1 protein or gene of said plant inactive, particularly during drought.
- In another embodiment, the present invention provides a method of improving seed development in drought conditions comprising inhibiting the activity of a G-protein, such as an RGA1 protein during drought conditions.
- In another embodiment, the present invention provides a hybrid plant, line or variety, wherein said plant hybrid, line or variety comprises germplasm comprising one or more mutations in a G-protein encoding gene, or the RGA1 gene such that the G-protein or RGA1 protein is inactive during drought conditions. This can be by use of a naturally occurring mutation such as the d1 mutant, or preferably by introduction an expression cassette to said plant designed to reduce activity of the RGA1 or other related G-protein gene either constitutively or under drought conditions.
- In some embodiments, said plant hybrid, line or variety is created by introgression of a plant germplasm that comprises said one or more modifications for rendering a G-protein or RGA1 gene or its encoded protein product inactive. In some embodiments, said plant hybrid, line or variety is created by incorporation of a heterologous genetic construct comprising designed to inhibit or otherwise decrease G-protein activity, particularly during drought conditions.
- In another embodiment, the present invention provides a method for producing a plant hybrid, line or variety resistant to drought conditions and with improved seed production compared to a wild type plant under the same conditions comprising identifying a germplasm with a null G-protein or RGA1 modification, and introducing said germplasm into an elite plant hybrid, line or variety. In some embodiments, said introducing of said germplasm into said elite hybrid, line or variety is by introgression. In some embodiments, said introducing of said germplasm into said elite plant hybrid, line or variety is by introduction of a heterologous genetic construct.
- In yet another embodiment, the present invention provides a plant hybrid, line or variety wherein the germplasm of said hybrid, line or variety confers drought tolerance with improved seed production.
- In yet another embodiment, the present invention provides a method for identifying plant lines resistant to drought comprising supplying a nucleic acid sample from a plant, providing amplification primers for amplifying a region of a plant corresponding to an G-protein or RGA1 nucleic acid sample, applying said amplification primers to said nucleic acid sample such that amplification of said region of said G-protein gene occurs, and identifying plants resistant to drought based on the presence of one or more null mutations in said amplified nucleic acid sample.
- In still another embodiment, the present invention provides for seeds wherein said germplasm of said seeds comprises a modified G-protein or RGA1 gene such that said mutation confers improved seed production under drought conditions.
- In some embodiments, the present invention provides for a plant that comprises a heterologous nucleotide sequence that is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the RGA1 gene of SEQ ID NO: 1, 2, 5, or 6. The invention also includes RGA1 polypeptides including the amino acid sequences of SEQ ID NO:3 or 4, conservatively modified variants which retain activity or loss of activity thereof as applicable.
- In one embodiment, the present invention provides a method of producing plant seed comprising crossing a plant comprising a genetically modified G-protein or a genetically modified RGA1 gene with itself or a second plant and collecting said seed from said cross. In some embodiments, the methods for producing said seed comprises planting a parent seed line wherein said parent seed line comprises a germplasm confers a decrease in activity or a G-protein or RGA1 mutation with a parent pollinator plant line wherein said pollinator and/or seed line germplasm comprises a germplasm that confers a modified G-protein or RGA1 protein, growing said parent seed and pollinator plants together, allowing for the said parent seed plants to be pollinated by said parent pollinator plants, and harvesting the seed that results from said pollination.
- In yet another embodiment, the invention provides for genetically modified plants incorporating a heterologous nucleotide construct encoding a RGA1 gene such as SEQ ID NOS: 1, 2, 5, or 6 operably linked to regulatory sequences such as expression cassettes, inhibition constructs, plants, plant cells, and seeds. The genetically modified plants, plant cells, and seeds of the invention may exhibit phenotypic changes, such as modulated RGA1 activity, particularly during drought conditions.
- Methods are provided for reducing or eliminating the activity of a G-protein or RGA1 polypeptide in a plant, comprising introducing into the plant a selected polynucleotide. In specific methods, providing the polynucleotide decreases the level of RGA1 or related G-protein in the plant.
- Methods are also provided for increasing the level of a modified RGA1 or G-protein polypeptide in a plant either constitutively or at specifically regulated times and tissues comprising introducing into the plant a selected polynucleotide with appropriate regulatory elements. In specific methods, expression of the modified RGA1 or G-protein polynucleotide improves the plants' tolerance to drought.
- Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range and include each integer within the defined range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. Unless otherwise provided for, software, electrical, and electronics terms as used herein are as defined in The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th Edition, 1993). The terms defined below are more fully defined by reference to the specification as a whole.
- By “amplified” is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Canteen, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
- As used herein, “chromosomal region” includes reference to a length of a chromosome that may be measured by reference to the linear segment of DNA that it comprises. The chromosomal region can be defined by reference to two unique DNA sequences, i.e., markers.
- The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids which encode identical or conservatively modified variants of the amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations” and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine; and UGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide of the present invention, is implicit in each described polypeptide sequence and is within the scope of the present invention.
- As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Thus, any number of amino acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for its native substrate. Conservative substitution tables providing functionally similar amino acids are well known in the art.
- The following six groups each contain amino acids that are conservative substitutions for one another:
- 1) Alanine (A), Serine (S), Threonine (T);
- 2) Aspartic acid (D), Glutamic acid (E);
- 3) Asparagine (N), Glutamine (Q);
- 4) Arginine (R), Lysine (K);
- 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
- 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
- See also, Creighton (1984) Proteins W.H. Freeman and Company.
- By “encoding” or “encoded”, with respect to a specified nucleic acid, is meant comprising the information for translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the “universal” genetic code. However, variants of the universal code, such as are present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum, or the ciliate Macronucleus, may be used when the nucleic acid is expressed therein.
- When the nucleic acid is prepared or altered synthetically, advantage can be taken of known codon preferences of the intended host where the nucleic acid is to be expressed. For example, although nucleic acid sequences of the present invention may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17:477-498 (1989)).
- As used herein “full-length sequence” in reference to a specified polynucleotide or its encoded protein means having the entire amino acid sequence of, a native (non-synthetic), endogenous, biologically active form of the specified protein. Methods to determine whether a sequence is full-length are well known in the art including such exemplary techniques as northern or western blots, primer extensions, S1 protection, and ribonuclease protection. See, e.g., Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to known full-length homologous (orthologous and/or paralogous) sequences can also be used to identify full-length sequences of the present invention. Additionally, consensus sequences typically present at the 5′ and 3′ untranslated regions of mRNA aid in the identification of a polynucleotide as full-length. For example, the consensus sequence ANNNNAUGG, where the underlined codon represents the N-terminal methionine, aids in determining whether the polynucleotide has a complete 5′ end. Consensus sequences at the 3′ end, such as polyadenylation sequences, aid in determining whether the polynucleotide has a complete 3′ end.
- As used herein, “heterologous” in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous structural gene is from a species different from that from which the structural gene was derived, or, if from the same species, one or both are substantially modified from their original form. A heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
- By “host cell” is meant a cell which contains a vector and supports the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells. Preferably, host cells are monocotyledonous or dicotyledonous plant cells. A particularly preferred monocotyledonous host cell is a maize host cell.
- The term “hybridization complex” includes reference to a duplex nucleic acid structure formed by two single-stranded nucleic acid sequences selectively hybridized with each other.
- The term “introduced” in the context of inserting a nucleic acid into a cell, means “transfection” or “transformation” or “transduction” and includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid may be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).
- The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components that normally accompany or interact with it as found in its naturally occurring environment. The isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically (non-naturally) altered by deliberate human intervention to a composition and/or placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment. The alteration to yield the synthetic material can be performed on the material within or removed from its natural state. For example, a naturally occurring nucleic acid becomes an isolated nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by means of human intervention performed within the cell from which it originates. See, e.g., Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868. Likewise, a naturally occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced by non-naturally occurring means to a locus of the genome not native to that nucleic acid. Nucleic acids which are “isolated” as defined herein are also referred to as “heterologous” nucleic acids.
- As used herein, “localized within the chromosomal region defined by and including” with respect to particular markers includes reference to a contiguous length of a chromosome delimited by and including the stated markers.
- As used herein, “marker” includes reference to a locus on a chromosome that serves to identify a unique position on the chromosome. A “polymorphic marker” includes reference to a marker which appears in multiple forms (alleles) such that different forms of the marker, when they are present in a homologous pair, allow transmission of each of the chromosomes of that pair to be followed. A genotype may be defined by use of one or a plurality of markers.
- By “nucleic acid library” is meant a collection of isolated DNA or cDNA molecules which comprise and substantially represent the entire transcribed fraction of a genome of a specified organism. Construction of exemplary nucleic acid libraries, such as genomic and cDNA libraries, is taught in standard molecular biology references such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994).
- As used herein, “operably linked” includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, “operably linked” means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
- As used herein, the term “plant” can include reference to whole plants, plant parts or organs (e.g., leaves, stems, roots, etc.), plant cells, seeds and progeny of same. Plant cell, as used herein, further includes, without limitation, cells obtained from or found in: seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant cells can also be understood to include modified cells, such as protoplasts, obtained from the aforementioned tissues. The class of plants which can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants. Particularly preferred plants include maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet.
- As used herein, “polynucleotide” includes reference to a deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons as “polynucleotides” as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including among other things, simple and complex cells.
- The terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids. The terms “polypeptide”, “peptide” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. It will be appreciated, as is well known and as noted above, that polypeptides are not entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Further, this invention contemplates the use of both the methionine-containing and the methionine-less amino terminal variants of the protein of the invention.
- As used herein “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds. Such promoters are referred to as “tissue preferred”. Promoters which initiate transcription only in certain tissue are referred to as “tissue specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may affect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most environmental conditions.
- As used herein “recombinant” includes reference to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found in identical form within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all as a result of deliberate human intervention. The term “recombinant” as used herein does not encompass the alteration of the cell or vector by naturally occurring events (e.g., spontaneous mutation, natural transformation/transduction/transposition) such as those occurring without deliberate human intervention.
- As used herein, a “recombinant expression cassette” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, nucleic acids to be transcribed, and a promoter.
- The terms “residue” or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass non-natural analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.
- The term “selectively hybridizes” includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, preferably 90% sequence identity, and most preferably 100% sequence identity (i.e., complementary) with each other.
- The term “stringent conditions” or “stringent hybridization conditions” includes reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and can be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length.
- Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 50° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. for 20 minutes.
- Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with 90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acids Probes, Part I,
Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995). In general a high stringency wash is 2×15 min in 0.5×SSC containing 0.1% SDS at 65° C. - As used herein, “genetically modified plant” includes reference to a plant which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. “Transgenic” is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term “transgenic” or “genetically modified” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
- As used herein, “vector” includes reference to a nucleic acid used in transfection of a host cell and into which can be inserted a polynucleotide. Vectors are often replicons. Expression vectors permit transcription of a nucleic acid inserted therein.
- The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence”, (b) “comparison window”, (c) “sequence identity”, (d) “percentage of sequence identity”, and (e) “substantial identity”.
- (a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
- (b) As used herein, “comparison window” includes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence may be compared to a reference sequence and wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence, a gap penalty is typically introduced and is subtracted from the number of matches.
- Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA; the CLUSTAL program is well described by Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS 5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24:307-331 (1994). The BLAST family of programs which can be used for database similarity searches includes: BLASTN for nucleotide query sequences against nucleotide database sequences; BLASTX for nucleotide query sequences against protein database sequences; BLASTP for protein query sequences against protein database sequences; TBLASTN for protein query sequences against nucleotide database sequences; and TBLASTX for nucleotide query sequences against nucleotide database sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
- Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters. Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information World Wide Web at ncbi.nih.gov. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).
- In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
- BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences which may be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions may be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexity filters can be employed alone or in combination.
- (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
- (d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
- (e) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 60%, or preferably at least 70%, 80%, 90%, and most preferably at least 95%.
- Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. However, nucleic acids which do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid sequence encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid sequence.
- (e) The terms “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70% sequence identity to a reference sequence, preferably 80%, or preferably 85%, most preferably at least 90% or 95% sequence identity to the reference sequence over a specified comparison window. Optionally, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides which are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes.
- The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
-
FIG. 1 shows the effects of water stress on wild-type and d1 plants during flowering and grain filling; mutant plants remain dark green and healthy while wild-type plants dry out and have a few living leaves under severe drought. The three watering conditions were: well-watered (100% soil relative water content), medium-water (45% soil relative water content), and low water (30% soil relative water content) conditions. Photographs were taken 140-160 days after emergence. -
FIG. 2 shows leaf temperatures on plants 165 days after emergence were measured by infrared thermography using a FLIR T620 Thermal Imaging Camera (FLIR Systems, USA). In order to obtain reliable comparisons, images were obtained over the shortest time interval possible; approximately 15 minutes total. Pseudo-colored temperature scales are indicated directly on the photographs. -
FIG. 3 shows that net photosynthesis and stomatal conductance measured on leaves between 120 and 130 days after emergence, using a Li-Cor 6400 Portable Photosynthesis System. Light intensity was 500 μmol m−2s−1. -
FIG. 4 shows the relationship between stomatal conductance and transpiration, measured under ambient conditions of 500 μmol m−2s−1 light intensity and 30° C. temperature using a Li-Cor 6400 Portable Photosynthesis System. Measurements were taken on seedlings at 40 days after emergence. Each data point represents an individual plant. Lines depicted are regression lines for each genotype; ANCOVA analysis demonstrated significant differences (P<0.05) in the slopes. -
FIG. 5 shows ψleaf measurements, which were made using a Scholander pressure chamber. Measurements were taken on the flag leaf of the primary tiller of plants 120-130 days after emergence. -
FIG. 6 is a graph showing the percentage of stalks flowering at the end of the reproductive phase in high water, medium water, and low water conditions. -
FIG. 7 shows the average number of panicles produced through time in d1 and wild-type plants. -
FIG. 8 shows the percentage of grain viability at the end of the reproductive cycle under high water, medium water and low water conditions. Data were obtained from 6 panicles randomly selected for analysis from 6 different plants of each genotype and water treatment combination, measured at the end of the life cycle. -
FIG. 9 is graph of yield of d1 and WT in well watered, medium water and low water conditions. -
FIG. 10 shows net photosynthesis and stomatal conductance at 1500 μmol m−2s−1 light measured on the flag leaf of the primary tiller at 60 days after emergence using a Li-Cor 6400 Portable Photosynthesis System. -
FIG. 11 shows that high planting density compromises plant growth and survival (as indicated by plant height) earlier in wild-type than in d1 plants. -
FIGS. 12A-B is a sequence alignment of the coding region of rgal and the d1 allele. (SEQ ID NO: 1 and 2 respectively). -
FIG. 13 is a sequence alignment of the encoded proteins of rgal and the d1 allele. (SEQ ID NO: 3 and 4 respectively). -
FIGS. 14A-D is a sequence alignment of the genomic region of rgal and the d1 allele. (SEQ ID NO: 5 and 6 respectively). - The rice dwarf mutant, d1, contains a non-functional RGA1 gene, encoding the GTP-binding α-subunit of the heterotrimeric G protein. This mutant was originally isolated as a spontaneous mutant with reduced height and shorter, erect, thicker, broad, dark green leaves, compact panicles, and short, round grains. Applicants have identified that a non-functional RGA1 gene or other related G-proteins can be used to create higher seed production and yield under drought conditions.
- Thus the present invention provides for compositions and methods for producing plants that have higher yield in drought conditions by manipulating the G-proteins such as the RGA1 gene or protein. In particular, the present invention provides for plants, varieties, lines, and hybrids, as well as plant tissues and plant seeds that contain modified G-protein activity, particularly RGA1 activity to engineer drought tolerance and improved seed production in plants.
- In one embodiment, the present invention provides for one or more plants whose germplasm has been modified to render a G-protein, particularly the RGA1 gene or its gene product inactive. Moreover, in further embodiments the invention relates to the offspring (e.g., F1, F2, F3, etc.) of a cross of said plant wherein the germplasm of said offspring has the same mutation as the parent plant. Therefore, embodiments of the present invention provide for plant varieties/hybrids whose germplasm contains a mutation, such that the phenotype of the plants is non-functional RGA1 gene activity in drought conditions. In some embodiments, said offspring (e.g., F1, F2, F3, etc.) are the result of a cross between elite lines, at least one of which contains a germplasm comprising a mutation that renders the G-protein such as RGA1 protein or gene of said plant inactive, particularly during drought.
- In another embodiment, the present invention provides a method of improving seed development in drought conditions comprising inhibiting the activity of a G-protein, such as RGA1 protein during drought conditions.
- In another embodiment, the present invention provides a hybrid plant, line or variety, wherein said plant hybrid, line or variety comprises germplasm comprising one or more mutations in a G-protein encoding gene, or the RGA1 gene such that the G-protein or RGA1 protein is inactive during drought conditions. This can be by use of a naturally occurring mutation such as the d1 mutant, or preferably by introduction an expression cassette to said plant designed to reduce activity of the RGA1 or other related G-protein gene either constitutively or under drought conditions.
- In some embodiments, said plant hybrid, line or variety is created by introgression of a plant germplasm that comprises said one or more modifications for rendering a G-protein or RGA1 gene or its encoded protein product inactive. In some embodiments, said plant hybrid, line or variety is created by incorporation of a heterologous genetic construct comprising designed to inhibit or otherwise decrease G-protein activity, particularly during drought conditions.
- In another embodiment, the present invention provides a method for producing a plant hybrid, line or variety resistant to drought conditions and with improved seed production compared to a wild type plant under the same conditions comprising identifying a germplasm with a null G-protein or RGA1 modification, and introducing said germplasm into an elite plant hybrid, line or variety. In some embodiments, said introducing of said germplasm into said elite hybrid, line or variety is by introgression. In some embodiments, said introducing of said germplasm into said elite plant hybrid, line or variety is by introduction of a heterologous genetic construct.
- In yet another embodiment, the present invention provides a plant hybrid, line or variety wherein the germplasm of said hybrid, line or variety comprises drought tolerance with improved seed production.
- In yet another embodiment, the present invention provides a method for identifying plant lines resistant to drought comprising supplying a nucleic acid sample from a plant, providing amplification primers for amplifying a region of a plant corresponding to a G-protein or RGA1 nucleic acid sample, applying said amplification primers to said nucleic acid sample such that amplification of said region of said G-protein gene occurs, and identifying plants resistant to drought based on the presence of one or more null mutations in said amplified nucleic acid sample.
- In still another embodiment, the present invention provides for seeds wherein said germplasm of said seeds comprises a modified G-protein or RGA1 gene such that said mutation confers improved seed production under drought conditions.
- In some embodiments, the present invention provides for a plant that comprises a heterologous nucleotide sequence that is at least 70% homologous, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 95% homologous, at least 97% homologous, or at least 99% homologous to the RGA1 gene of SEQ ID NO:1, 2, 5, or 6. The invention also includes RGA1 polypeptides including the amino acid sequences of SEQ ID NO: 3 or 4, conservatively modified variants which retain activity or loss of activity thereof as applicable.
- In one embodiment, the present invention provides a method of producing plant seed comprising crossing a plant comprising a genetically modified G-protein or RGA1 gene with itself or a second plant and collecting said seed from said cross. In some embodiments, the methods for producing said seed comprises planting a parent seed line wherein said parent seed line comprises a germplasm confers a decrease in activity or a G-protein or RGA1 mutation with a parent pollinator plant line wherein said pollinator and/or seed line germplasm comprises a germplasm that confers a modified G-protein of RGA1 protein, growing said parent seed and pollinator plants together, allowing for the said parent seed plants to be pollinated by said parent pollinator plants, and harvesting the seed that results from said pollination.
- In yet another embodiment, the invention provides for genetically modified plants incorporating a heterologous nucleotide construct encoding a RGA1 gene such as SEQ ID NOS: 1, 2, 5, or 6 operably linked to regulatory sequences such as expression cassettes, inhibition constructs, plants, plant cells, and seeds. The genetically modified plants, plant cells, and seeds of the invention may exhibit phenotypic changes, such as modulated RGA1 activity, particularly during drought conditions.
- Methods are provided for reducing or eliminating the activity of a G-protein or RGA1 polypeptide in a plant, comprising introducing into the plant a selected polynucleotide. In specific methods, providing the polynucleotide decreases the level of RGA1 or related G-protein in the plant.
- Methods are also provided for increasing the level of a modified RGA1 or G-protein polypeptide in a plant either constitutively or at specifically regulated times and tissues comprising introducing into the plant a selected polynucleotide with appropriate regulatory elements. In specific methods, expression of the modified RGA1 or G-protein polynucleotide improves the plant's tolerance to drought.
- The following is a non-limiting general overview of molecular biology techniques which may be used in performing the methods of the invention.
- The constructs, promoters or control systems used in the methods of the invention may include a tissue specific promoter, an inducible promoter or a constitutive promoter.
- A large number of suitable promoter systems are available. For example one constitutive promoter useful for the invention is the cauliflower mosaic virus (CaMV) 35S. It has been shown to be highly active in many plant organs and during many stages of development when integrated into the genome of transgenic plants and has been shown to confer expression in protoplasts of both dicots and monocots.
- Organ-specific promoters are also well known. For example, the E8 promoter is only transcriptionally activated during tomato fruit ripening, and can be used to target gene expression in ripening tomato fruit (Deikman and Fischer, EMBO J. (1988) 7:3315; Giovannoni et al., The Plant Cell (1989) 1:53). The activity of the E8 promoter is not limited to tomato fruit, but is thought to be compatible with any system wherein ethylene activates biological processes. Similarly the Lipoxegenase (“the LOX gene”) is a fruit specific promoter.
- Other fruit specific promoters are the 1.45 promoter fragment disclosed in Bird, et al., Plant Mol. Bio., pp 651-663(1988) and the polygalacturonase promoter from tomato disclosed in U.S. Pat. No. 5,413,937 to Bridges et al.
- Leaf specific promoters include as the AS-1 promoter disclosed in U.S. Pat. No. 5,256,558 to Coruzzi and the RBCS-3A promoter isolated from pea the RBCS-3A gene disclosed in U.S. Pat. No. 5,023,179 to Lam et al.
- And finally root specific promoters include the CamV 35S promoter disclosed in U.S. Pat. No. 391,725 to Coruzzi et al; the RB7 promoter disclosed in U.S. Pat. No. 5,459,252 to Conking et al. and the promoter isolated from Brassica napus disclosed in U.S. Pat. No. 5,401,836 to Bazczynski et al. which give root specific expression.
- Other examples of promoters include maternal tissue promoters such as seed coat, pericarp and ovule. Promoters highly expressed early in endosperm development are most effective in this application. Of particular interest is the promoter from the a′ subunit of the soybean β-conglycinin gene [Walling et al., Proc. Natl. Acad. Sci. USA 83:2123-2127 (1986)] which is expressed early in seed development in the endosperm and the embryo.
- Further seed specific promoters include the Napin promoter described in U.S. Pat. No. 5,110,728 to Calgene, which describes and discloses the use of the napin promoter in directing the expression to seed tissue of an acyl carrier protein to enhance seed oil production; the DC3 promoter from carrots which is early to mid embryo specific and is disclosed at Plant Physiology, October 1992 100(2) p. 576-581, “Hormonal and Environmental Regulation of the Carrot Lea-
class Gene Dc 3, and Plant Mol. Biol., April 1992, 18(6) p. 1049-1063, “Transcriptional Regulation of a Seed Specific Carrot Gene, DC 8”: the phaseolin promoter described in U.S. Pat. No. 5,504,200 to Mycogen which discloses the gene sequence and regulatory regions for phaseolin, a protein isolated from P. vulgaris which is expressed only while the seed is developing within the pod, and only in tissues involved in seed generation. - Other organ-specific promoters appropriate for a desired target organ can be isolated using known procedures. These control sequences are generally associated with genes uniquely expressed in the desired organ. In a typical higher plant, each organ has thousands of mRNAs that are absent from other organ systems (reviewed in Goldberg, Phil, Trans. R. Soc. London (1986) B314-343. mRNAs are first isolated to obtain suitable probes for retrieval of the appropriate genomic sequence which retains the presence of the natively associated control sequences. An example of the use of techniques to obtain the cDNA associated with mRNA specific to avocado fruit is found in Christoffersen et al., Plant Molecular Biology (1984) 3:385. Briefly, mRNA was isolated from ripening avocado fruit and used to make a cDNA library. Clones in the library were identified that hybridized with labeled RNA isolated from ripening avocado fruit, but that did not hybridize with labeled RNAs isolated from unripe avocado fruit. Many of these clones represent mRNAs encoded by genes that are transcriptionally activated at the onset of avocado fruit ripening.
- Another very important method that can be used to identify cell type specific promoters that allow identification of genes expressed even in a single cell is enhancer detection (O'Kane, C., and Gehring, W. J. (1987), “Detection in situ of genomic regulatory elements in Drosophila”, Proc. Natl. Acad. Sci. USA, 84, 9123-9127). This method was first developed in Drosophila and rapidly adapted to mice and plants (Wilson, C., Pearson, R. K., Bellen, H. J., O'Kane, C. J., Grossniklaus, U., and Gehring, W. J. (1989)), “P-element-mediated enhancer detection: an efficient method for isolating and characterizing developmentally regulated genes in Drosophila”, Genes & Dev., 3, 1301-1313; Skarnes, W. C. (1990), “Entrapment vectors: a new tool for mammalian genetics”, Biotechnology, 8, 827-831; Topping, J. F., Wei, W., and Lindsey, K. (1991), “Functional tagging of regulatory elements in the plant genome”, Development, 112, 1009-1019; Sundaresan, V., Springer, P. S., Volpe, T., Haward, S., Jones, J. D. G., Dean, C., Ma, H., and Martienssen, R. A., (1995), “Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements”, Genes & Dev., 9, 1797-1810).
- The promoter used in the method of the invention may be an inducible promoter. An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of a DNA sequence in response to an inducer. In the absence of an inducer, the DNA sequence will not be transcribed. Typically, the protein factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer may be a chemical agent such as a protein, metabolite (sugar, alcohol etc.), a growth regulator, herbicide, or a phenolic compound or a physiological stress imposed directly by heat, salt, toxic elements etc. or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell such as by spraying, watering, heating, or similar methods. Examples of inducible promoters include the inducible 70 kD heat shock promoter of (Freeling, M., Bennet, D. C.,
Maize ADN 1, Ann. Rev. of Genetics, 19:297-323) and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, R. T., et al., Miflin, B. J., Ed. Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3, p. 384-438, Oxford University Press, Oxford 1986) or the Lex A promoter which is triggered with chemical treatment and is available through Ligand Pharmaceuticals. The inducible promoter may be in an induced state throughout seed formation or at least for a period which corresponds to the transcription of the DNA sequence of the recombinant DNA molecule(s). - Another example of an inducible promoter is the chemically inducible gene promoter sequence isolated from a 27 kD subunit of the maize glutathione-S-transferase (GST II) gene. Two of the inducers for this promoter are N,N-diallyl-2,2-dichloroacetamide (common name: dichloramid) or benzyl-=2-chloro-4-(trifluoromethyl)-5-thiazolecarboxylate (common name: flurazole). In addition, a number of other potential inducers may be used with this promoter as described in published PCT Application No. PCT/GB90/00110 by ICI.
- Another example of an inducible promoter is the light inducible chlorophyll a/b binding protein (CAB) promoter, also described in published PCT Application No. PCT/GB90/00110 by ICI.
- Inducible promoters have also been described in published Application No. EP89/103888.7 by Ciba-Geigy. In this application, a number of inducible promoters are identified, including the PR protein genes, especially the tobacco PR protein genes, such as PR-1a, PR-1b, PR-1c, PR-1, PR-A, PR-S, the cucumber chitinase gene, and the acidic and basic tobacco beta-1,3-glucanase genes. There are numerous potential inducers for these promoters, as described in Application No. EP89/103888.7.
- The preferred promoters may be used in conjunction with naturally occurring flanking coding or transcribed sequences of the feronia regulatory genes or with any other coding or transcribed sequence that is critical to pollen tube formation and/or fertilization.
- It may also be desirable to include some intron sequences in the promoter constructs since the inclusion of intron sequences in the coding region may result in enhanced expression and specificity. Thus, it may be advantageous to join the DNA sequences to be expressed to a promoter sequence that contains the first intron and exon sequences of a polypeptide which is unique to cells/tissues of a plant critical to female gametophyte development and/or function.
- Additionally, regions of one promoter may be joined to regions from a different promoter in order to obtain the desired promoter activity resulting in a chimeric promoter. Synthetic promoters which regulate gene expression may also be used.
- The expression system may be further optimized by employing supplemental elements such as transcription terminators and/or enhancer elements.
- In addition to a promoter sequence, an expression cassette or construct should also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region or polyadenylation signal may be obtained from the same gene as the promoter sequence or may be obtained from different genes. Polyadenylation sequences include, but are not limited to the Agrobacterium octopine synthase signal (Gielen et al., EMBO J. (1984) 3:835-846) or the nopaline synthase signal (Depicker et al., Mol. and Appl. Genet. (1982) 1:561-573).
- Recombinant DNA molecules containing any of the DNA sequences and promoters described herein may additionally contain selection marker genes which encode a selection gene product which confer on a plant cell resistance to a chemical agent or physiological stress, or confers a distinguishable phenotypic characteristic to the cells such that plant cells transformed with the recombinant DNA molecule may be easily selected using a selective agent. One such selection marker gene is neomycin phosphotransferase (NPT II) which confers resistance to kanamycin and the antibiotic G-418. Cells transformed with this selection marker gene may be selected for by assaying for the presence in vitro of phosphorylation of kanamycin using techniques described in the literature or by testing for the presence of the mRNA coding for the NPT II gene by Northern blot analysis in RNA from the tissue of the transformed plant. Polymerase chain reactions are also used to identify the presence of a transgene or expression using reverse transcriptase PCR amplification to monitor expression and PCR on genomic DNA. Other commonly used selection markers include the ampicillin resistance gene, the tetracycline resistance and the hygromycin resistance gene. Transformed plant cells thus selected can be induced to differentiate into plant structures which will eventually yield whole plants. It is to be understood that a selection marker gene may also be native to a plant.
- Numerous methods for plant transformation have been developed, including biological and physical, plant transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.
- A. Agrobacterium-Mediated Transformation
- One method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., Science 227: 1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant. Sci. 10: 1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al., supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8: 238 (1989). See also, U.S. Pat. No. 5,563,055, (Townsend and Thomas), issued Oct. 8, 1996.
- B. Direct Gene Transfer
- Several methods of plant transformation, collectively referred to as direct gene transfer, have been developed as an alternative to Agrobacterium-mediated transformation. A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 μm. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Sanford et al., Part. Sci. Technol. 5: 27 (1987), Sanford, J. C., Trends Biotech. 6: 299 (1988), Klein et al., Bio/Technology 6: 559-563 (1988), Sanford, J. C., Physiol Plant 79: 206 (1990), Klein et al., Biotechnology 10: 268 (1992). See also U.S. Pat. No. 5,015,580 (Christou, et al), issued May 14, 1991; U.S. Pat. No. 5,322,783 (Tomes, et al.), issued Jun. 21, 1994.
- Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9: 996 (1991). Alternatively, liposome or spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al.,
EMBO 1, 4: 2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84: 3962 (1987). Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet. 199: 161 (1985) and Draper et al., Plant Cell Physiol. 23: 451 (1982). Electroporation of protoplasts and whole cells and tissues have also been described. Donn et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4: 1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24: 51-61 (1994). - Following transformation of target tissues, expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.
- It is often desirable to have the DNA sequence in homozygous state which may require more than one transformation event to create a parental line, requiring transformation with a first and second recombinant DNA molecule both of which encode the same gene product. It is further contemplated in some of the embodiments of the process of the invention that a plant cell be transformed with a recombinant DNA molecule containing at least two DNA sequences or be transformed with more than one recombinant DNA molecule. The DNA sequences or recombinant DNA molecules in such embodiments may be physically linked, by being in the same vector, or physically separate on different vectors. A cell may be simultaneously transformed with more than one vector provided that each vector has a unique selection marker gene. Alternatively, a cell may be transformed with more than one vector sequentially allowing an intermediate regeneration step after transformation with the first vector. Further, it may be possible to perform a sexual cross between individual plants or plant lines containing different DNA sequences or recombinant DNA molecules preferably the DNA sequences or the recombinant molecules are linked or located on the same chromosome, and then selecting from the progeny of the cross, plants containing both DNA sequences or recombinant DNA molecules.
- Expression of recombinant DNA molecules containing the DNA sequences and promoters described herein in transformed plant cells may be monitored using Northern blot techniques and/or Southern blot techniques known to those of skill in the art.
- The transformed cells may then be regenerated into a transgenic plant. The regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner.
- After the expression or inhibition cassette is stably incorporated into regenerated transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. It may be useful to generate a number of individual transformed plants with any recombinant construct in order to recover plants free from any position effects. It may also be preferable to select plants that contain more than one copy of the introduced recombinant DNA molecule such that high levels of expression of the recombinant molecule are obtained.
- As indicated above, it may be desirable to produce plant lines which are homozygous for a particular gene. In some species this is accomplished rather easily by the use of another culture or isolated microspore culture. This is especially true for the oil seed crop Brassica napus (Keller and Armstrong, Z. Pflanzenzucht 80:100-108, 1978). By using these techniques, it is possible to produce a haploid line that carries the inserted gene and then to double the chromosome number either spontaneously or by the use of colchicine. This gives rise to a plant that is homozygous for the inserted gene, which can be easily assayed for if the inserted gene carries with it a suitable selection marker gene for detection of plants carrying that gene. Alternatively, plants may be self-fertilized, leading to the production of a mixture of seed that consists of, in the simplest case, three types, homozygous (25%), heterozygous (50%) and null (25%) for the inserted gene. Although it is relatively easy to score null plants from those that contain the gene, it is possible in practice to score the homozygous from heterozygous plants by Southern blot analysis in which careful attention is paid to the loading of exactly equivalent amounts of DNA from the mixed population, and scoring heterozygotes by the intensity of the signal from a probe specific for the inserted gene. It is advisable to verify the results of the Southern blot analysis by allowing each independent transformant to self-fertilize, since additional evidence for homozygosity can be obtained by the simple fact that if the plant was homozygous for the inserted gene, all of the subsequent plants from the selfed seed will contain the gene, while if the plant was heterozygous for the gene, the generation grown from the selfed seed will contain null plants. Therefore, with simple selfing one can easily select homozygous plant lines that can also be confirmed by southern blot analysis.
- Creation of homozygous parental lines makes possible the production of hybrid plants and seeds which will contain a modified protein component. Transgenic homozygous parental lines are maintained with each parent containing either the first or second recombinant DNA sequence operably linked to a promoter. Also incorporated in this scheme are the advantages of growing a hybrid crop, including the combining of more valuable traits and hybrid vigor.
- The nucleotide constructs of the invention also encompass nucleotide constructs that may be employed in methods for altering or mutating a genomic nucleotide sequence in an organism, including, but not limited to, chimeric vectors, chimeric mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides, self-complementary chimeric oligonucleotides, and recombinogenic oligonucleobases. Such nucleotide constructs and methods of use, such as, for example, chimeraplasty, are known in the art. Chimeraplasty involves the use of such nucleotide constructs to introduce site-specific changes into the sequence of genomic DNA within an organism. See, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984; all of which are herein incorporated by reference. See also, WO 98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc. Natl. Acad. Sci. USA 96:8774-8778; herein incorporated by reference.
- The present invention provides a method of genotyping a plant comprising a heterologous polynucleotide of the present invention. Genotyping provides a means of distinguishing homologs of a chromosome pair and can be used to differentiate segregants in a plant population. Molecular marker methods can be used for phylogenetic studies, characterizing genetic relationships among crop varieties, identifying crosses or somatic hybrids, localizing chromosomal segments affecting monogenic traits, map based cloning, and the study of quantitative inheritance. See, e.g., Clark, Ed., Plant Molecular Biology: A Laboratory Manual. Berlin, Springer Verlag, 1997.
Chapter 7. For molecular marker methods, see generally, “The DNA Revolution” in: Paterson, A. H., Genome Mapping in Plants (Austin, Tex., Academic Press/R. G. Landis Company, 1996) pp. 7-21. - The particular method of genotyping in the present invention may employ any number of molecular marker analytic techniques such as, but not limited to, restriction fragment length polymorphisms (RFLPs). RFLPs are the product of allelic differences between DNA restriction fragments resulting from nucleotide sequence variability. As is well known to those of skill in the art, RFLPs are typically detected by extraction of genomic DNA and digestion with a restriction enzyme. Generally, the resulting fragments are separated according to size and hybridized with a probe; single copy probes are preferred. Restriction fragments from homologous chromosomes are revealed.
- Differences in fragment size among alleles represent an RFLP. Thus, the present invention further provides a means to follow segregation of a gene or nucleic acid of the present invention as well as chromosomal sequences genetically linked to these genes or nucleic acids using such techniques as RFLP analysis. Linked chromosomal sequences are within 50 centiMorgans (cM), often within 40 or 30 cM, preferably within 20 or 10 cM, more preferably within 5, 3, 2, or 1 cM of a gene of the present invention.
- In the present invention, the nucleic acid probes employed for molecular marker mapping of plant nuclear genomes selectively hybridize, under selective hybridization conditions, to a gene encoding a polynucleotide of the present invention. In preferred embodiments, the probes are selected from polynucleotides of the present invention.
- Typically, these probes are cDNA probes or restriction-enzyme treated (e.g., Pst I) genomic clones. The length of the probes is discussed in greater detail, supra, but are typically at least 15 bases in length, more preferably at least 20, 25, 30, 35, 40, or 50 bases in length. Generally, however, the probes are less than about 1 kilobase in length. Preferably, the probes are single copy probes that hybridize to a unique locus in a haploid chromosome complement. Some exemplary restriction enzymes employed in RFLP mapping are EcoRI, EcoRv, and SstI. As used herein the term “restriction enzyme” includes reference to a composition that recognizes and, alone or in conjunction with another composition, cleaves at a specific nucleotide sequence.
- The method of detecting an RFLP comprises the steps of (a) digesting genomic DNA of a plant with a restriction enzyme; (b) hybridizing a nucleic acid probe, under selective hybridization conditions, to a sequence of a polynucleotide of the present of said genomic DNA; (c) detecting therefrom a RFLP. Other methods of differentiating polymorphic (allelic) variants of polynucleotides of the present invention can be had by utilizing molecular marker techniques well known to those of skill in the art including such techniques as: 1) single stranded conformation analysis (SSCA); 2) denaturing gradient gel electrophoresis (DGGE); 3) RNase protection assays; 4) allele-specific oligonucleotides (ASOs); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein; and 6) allele-specific PCR. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); and chemical mismatch cleavage (CMC). Thus, the present invention further provides a method of genotyping comprising the steps of contacting, under stringent hybridization conditions, a sample suspected of comprising a polynucleotide of the present invention with a nucleic acid probe. Generally, the sample is a plant sample; preferably, a sample suspected of comprising a maize polynucleotide of the present invention (e.g., gene, mRNA). The nucleic acid probe selectively hybridizes, under stringent conditions, to a subsequence of a polynucleotide of the present invention comprising a polymorphic marker. Selective hybridization of the nucleic acid probe to the polymorphic marker nucleic acid sequence yields a hybridization complex. Detection of the hybridization complex indicates the presence of that polymorphic marker in the sample. In preferred embodiments, the nucleic acid probe comprises a polynucleotide of the present invention.
- In general, translational efficiency has been found to be regulated by specific sequence elements in the 5′ non-coding or untranslated region (5′UTR) of the RNA. Positive sequence motifs include translational initiation consensus sequences (Kozak, Nucleic Acids Res. 15: 8125 (1987)) and the 7-methylguanosine cap structure (Drummond et al., Nucleic Acids Res. 13: 7375 (1985)). Negative elements include stable intramolecular 5′ UTR stem-loop structures (Muesing et al., Cell 48: 691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5 ‘UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8: 284 (1988)). Accordingly, the present invention provides 5’ and/or 3′ untranslated regions for modulation of translation of heterologous coding sequences.
- Further, the polypeptide-encoding segments of the polynucleotides of the present invention can be modified to alter codon usage. Altered codon usage can be employed to alter translational efficiency and/or to optimize the coding sequence for expression in a desired host such as to optimize the codon usage in a heterologous sequence for expression in maize. Codon usage in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages such as “Codon Preference” available from the University of Wisconsin Genetics Computer Group (see Devereaux et al., Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Thus, the present invention provides a codon usage frequency characteristic of the coding region of at least one of the polynucleotides of the present invention. The number of polynucleotides that can be used to determine a codon usage frequency can be any integer from 1 to the number of polynucleotides of the present invention as provided herein. Optionally, the polynucleotides will be full-length sequences. An exemplary number of sequences for statistical analysis can be at least 1, 5, 10, 20, 50, or 100.
- The present invention provides methods for sequence shuffling using polynucleotides of the present invention, and compositions resulting therefrom. Sequence shuffling is described in PCT publication No. WO 97/20078. See also, Zhang, J.-H., et al. Proc. Natl. Acad. Sci. USA 94: 4504-4509 (1997). Generally, sequence shuffling provides a means for generating libraries of polynucleotides having a desired characteristic which can be selected or screened for. Libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides which comprise sequence regions which have substantial sequence identity and can be homologously recombined in vitro or in vivo. The population of sequence-recombined polynucleotides comprises a subpopulation of polynucleotides which possess desired or advantageous characteristics and which can be selected by a suitable selection or screening method. The characteristics can be any property or attribute capable of being selected for or detected in a screening system, and may include properties of: an encoded protein, a transcriptional element, a sequence controlling transcription, RNA processing, RNA stability, chromatin conformation, translation, or other expression property of a gene or transgene, a replicative element, a protein-binding element, or the like, such as any feature which confers a selectable or detectable property. In some embodiments, the selected characteristic will be a decreased Km and/or increased KCat over the wild-type protein as provided herein. In other embodiments, a protein or polynucleotide generated from sequence shuffling will have a ligand binding affinity greater than the non-shuffled wild-type polynucleotide. The increase in such properties can be at least 110%, 120%, 130%, 140% or at least 150% of the wild-type value.
- Polynucleotides and polypeptides of the present invention further include those having: (a) a generic sequence of at least two homologous polynucleotides or polypeptides, respectively, of the present invention; and, (b) a consensus sequence of at least three homologous polynucleotides or polypeptides, respectively, of the present invention. The generic sequence of the present invention comprises each species of polypeptide or polynucleotide embraced by the generic polypeptide or polynucleotide sequence, respectively. The individual species encompassed by a polynucleotide having an amino acid or nucleic acid consensus sequence can be used to generate antibodies or produce nucleic acid probes or primers to screen for homologs in other species, genera, families, orders, classes, phyla, or kingdoms.
- Alternatively, a polynucleotide having a consensus sequence generated from orthologous genes can be used to identify or isolate orthologs of other taxa. Typically, a polynucleotide having a consensus sequence will be at least 25, 30, or 40 in nucleotides in length. As those of skill in the art are aware, a conservative amino acid substitution can be used for amino acids which differ amongst aligned sequences but are from the same conservative substitution group as discussed above. Optionally, no more than 1 or 2 conservative amino acids are substituted for each 10 amino acid length of consensus sequence.
- Similar sequences used for generation of a consensus or generic sequence include any number and combination of allelic variants of the same gene, orthologous, or paralogous sequences as provided herein. Optionally, similar sequences used in generating a consensus or generic sequence are identified using the BLAST algorithm's smallest sum probability (P(N)). Various suppliers of sequence-analysis software are listed in
chapter 7 of Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (Supplement 30). - A polynucleotide sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, or 0.001, and most preferably less than about 0.0001, or 0.00001. Similar polynucleotides can be aligned and a consensus or generic sequence generated using multiple sequence alignment software available from a number of commercial suppliers such as the Genetics Computer Group's (Madison, Wis.) PILEUP software, Vector NTI's (North Bethesda, Md.) ALIGNX, or Genecode's (Ann Arbor, Mich.) SEQUENCHER. Conveniently, default parameters of such software can be used to generate consensus or generic sequences.
- Reducing the Activity and/or Level of an RGA1 Polypeptide
- Methods are also provided to reduce or eliminate the activity of an RGA1 polypeptide by transforming a plant cell with an expression cassette that expresses a polynucleotide that inhibits the expression of the RGA1. The polynucleotide may inhibit the expression of the RGA1 directly, by preventing transcription or translation of the RGA1 messenger RNA, or indirectly, by encoding a polypeptide that inhibits the transcription or translation of an RGA1 gene encoding an RGA1 polypeptide. Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of the RGA1 polypeptide. Many methods may be used to reduce or eliminate the activity of an RGA1 polypeptide. In addition, more than one method may be used to reduce the activity of a single RGA1 polypeptide.
- 1. Polynucleotide-Based Methods:
- In some embodiments of the present invention, a plant is transformed with an expression cassette that is capable of expressing a polynucleotide that inhibits the expression of an RGA1 polypeptide of the invention. The term “expression” as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, for the purposes of the present invention, an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one RGA1 polypeptide is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one RGA1 polypeptide of the invention. The “expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the “expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.
- Examples of polynucleotides that inhibit the expression of an RGA1 polypeptide include sense Suppression/Cosuppression, where an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding an RGA1 polypeptide in the “sense” orientation and over expression of the RNA molecule can result in reduced expression of the native gene; Antisense Suppression where the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA encoding the RGA1 polypeptide and over expression of the antisense RNA molecule can result in reduced expression of the native gene; Double-Stranded RNA Interference, where a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA, Hairpin RNA Interference and Intron-Containing Hairpin RNA Interference.
- Where the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop region and a base-paired stem, Small Interfering RNA or Micro RNA, where the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA such as small interfering RNA or artificial micro RNAs.
- 2. Polypeptide-Based Inhibition of Gene Expression
- In one embodiment, the polynucleotide encodes a zinc finger protein that binds to a gene encoding an RGA1 polypeptide, resulting in reduced expression of the gene methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242, and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in U.S. Patent Publication No. 2003/0037355; each of which is herein incorporated by reference.
- 3. Polypeptide-Based Inhibition of Protein Activity
- In some embodiments of the invention, the polynucleotide encodes an antibody that binds to at least one RGA1 polypeptide and reduces the activity of the RGA1 polypeptide. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald, (2003) Nature Biotech. 21:35-36, incorporated herein by reference.
- 4. Gene Disruption
- In some embodiments of the present invention, the activity of an RGA1 polypeptide is reduced or eliminated by disrupting the gene encoding the RGA1 polypeptide. The gene encoding the RGA1 polypeptide may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing plants using random or targeted mutagenesis, and selecting for plants that have a desired trait or phenotype.
- In certain embodiments the nucleic acid sequences of the present invention can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired phenotype. For example, the polynucleotides of the present invention may be stacked with any other polynucleotides of the present invention, (SEQ ID NO: 1), or with other genes implicated in herbicide resistance. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409)); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122); and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased digestibility (e.g., modified storage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001)); and thioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001), the disclosures of which are herein incorporated by reference.
- The polynucleotides of the present invention can also be stacked with traits desirable for insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al (1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene and GAT gene)); and traits desirable for processing or process products such as high oil (U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics (U.S. Pat. No. 5,602,321); beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847), which facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the present invention with polynucleotides providing agronomic traits such as male sterility (see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (see, WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.
- These stacked combinations can be created by any method including, but not limited to, polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant.
- The transformed plants of the invention may be used in a plant breeding program. The goal of plant breeding is to combine, in a single variety or hybrid, various desirable traits. For field crops, these traits may include, for example, resistance to diseases and insects, tolerance to heat and drought, tolerance to high planting density, reduced time to crop maturity, greater yield, and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant height is desirable. Traditional plant breeding is an important tool in developing new and improved commercial crops. This invention encompasses methods for producing a plant by crossing a first parent plant with a second parent plant wherein one or both of the parent plants is a transformed plant according to the invention displaying drought tolerance as described herein.
- Plant breeding techniques known in the art and used in a plant breeding program include, but are not limited to, recurrent selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, doubled haploids, and transformation. Often combinations of these techniques are used.
- The development of hybrids in a plant breeding program requires, in general, the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. There are many analytical methods available to evaluate the result of a cross. The oldest and most traditional method of analysis is the observation of phenotypic traits. Alternatively, the genotype of a plant can be examined.
- A genetic trait which has been engineered into a particular plant using transformation techniques can be moved into another line using traditional breeding techniques that are well known in the plant breeding arts. For example, a backcrossing approach is commonly used to move a transgene from a transformed maize plant to an elite inbred line, and the resulting progeny would then comprise the transgene(s). Also, if an inbred line was used for the transformation, then the transgenic plants could be crossed to a different inbred in order to produce a transgenic hybrid plant. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
- The development of a hybrid in a plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, while different from each other, breed true and are highly uniform; and (3) crossing the selected inbred lines with different inbred lines to produce the hybrids. During the inbreeding process, the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid. An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a defined pair of inbreds will always be the same. Once the inbreds that give a superior hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parents is maintained.
- Transgenic plants of the present invention may be used to produce, e.g., a single cross hybrid, a three-way hybrid or a double cross hybrid. A single cross hybrid is produced when two inbred lines are crossed to produce the F1 progeny. A double cross hybrid is produced from four inbred lines crossed in pairs (A×B and C×D) and then the two F1 hybrids are crossed again (A×B)×(C×D). A three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A×B) and then the resulting F1 hybrid is crossed with the third inbred (A×B)×C. Much of the hybrid vigor and uniformity exhibited by F1 hybrids is lost in the next generation (F2). Consequently, seed produced by hybrids is consumed rather than planted.
- All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
- Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims. Thus, many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.
- The following examples are offered by way of illustration and not by way of limitation.
- The rice dwarf mutant, d1, contains a non-functional RGA1 gene, encoding the GTP-binding α-subunit of the heterotrimeric G protein. This mutant was originally isolated as a spontaneous mutant with reduced height and shorter, erect, thicker, broad, dark green leaves, compact panicles, and short, round grains. We have examined the physiological responses of the d1 mutant to mild and severe water limitation during both vegetative and reproductive development in comparison with its background line. The d1 plants present higher photosynthetic rates, stomatal conductance, and ψleaf than wild type during both mild and severe water limitation, and have a greater number and percentage of tillers producing panicles, with resulting higher reproductive yield. The d1 plants also perform better than wild-type plants under high planting densities.
-
FIG. 1 shows water stress during flowering and grain filling; mutant plants remain dark green and healthy while wild-type plants dry out and have a few living leaves under severe drought. The three watering conditions were: well-watered (100% soil relative water content), medium-water (45% soil relative water content), and low water (30% soil relative water content) conditions. Photographs were taken 140-160 days after emergence. -
FIG. 2 shows that d1 plants exhibit lower leaf temperatures than wild-type plants under all 3 watering conditions. Leaf temperatures on plants 165 days after emergence were measured by infrared thermography using a FLIR T620 Thermal Imaging Camera (FLIR Systems, USA). In order to obtain reliable comparisons, images were obtained over the shortest time interval possible; approximately 15 minutes total. Pseudo-colored temperature scales are indicated directly on the photographs. -
FIG. 3 shows that net photosynthesis and stomatal conductance are reduced more severely in wild-type plants than in d1 mutants under medium-water (45% soil relative water content) and low-water (30% soil relative water content) conditions, while these parameters are statistically identical under well-watered (100% soil relative water content) conditions. Parameters were measured on leaves between 120 and 130 days after emergence, using a Li-Cor 6400 Portable Photosynthesis System. Light intensity was 500 m−2s−1. -
FIG. 4 shows that d1 mutants have higher stomatal conductance but nevertheless lose less water (have lower transpiration rates) than wild-type plants. This is presumably due to the lower leaf temperatures of d1 plants as compared to wild-type (as shown inFIG. 3 ); lower leaf temperatures result in a reduced vapor pressure deficit, i.e. a reduced physical driving force for evaporative water loss. Measurements were made under ambient conditions of 500 μmol m−2s−1 light and 30° C. temperature using a Li-Cor 6400 Portable Photosynthesis System. Measurements were taken on seedlings at 40 days after emergence. Each data point represents an individual plant. Lines depicted are regression lines for each genotype; ANCOVA analysis demonstrated significant differences (P<0.05) in the slopes. -
FIG. 5 shows that the water potential (ψleaf) of d1 plants is higher than that of wild-type plants under medium-water and low-water conditions, and equal to that of wild-type plants under well-watered conditions (the three watering regimes were specified above). ψleaf measurements, made using a Scholander pressure chamber, were taken from the flag leaf of the primary tiller of plants 120-130 days after emergence. - Taken together,
FIGS. 3-5 show that d1 plants have improved water status and higher photosynthetic rates than wild-type plants under drought conditions. -
FIG. 6 is a graph showing the percentage of stalks flowering at the end of the reproductive phase in high water, low water and medium water conditions. One can see that water limitation decreases the number of stalks developing a flower head in wild-type plants but not in d1 mutants. -
FIG. 7 shows the average number of panicles produced through time in d1 and wild-type plants. Panicle production is greater in the d1 mutant than in wild-type plants. Water-limited conditions result in a pronounced reduction in the average number of panicles produced by wild-type plants but do not affect average panicle production in d1 plants. -
FIG. 8 shows drought-induced grain abortion per panicle in wild-type and d1 plants under the three watering regimes. The data are from 6 panicles randomly selected for analysis from 6 different plants of each genotype and water treatment combination, measured at the end of the life cycle. - Taken together, the above 8 figures show the basis for the increased grain yield by d1 plants as compared to wild-type plants under drought conditions:
FIG. 9 shows that estimated grain yield per plant is statistically equal in wild-type and mutant d1 plants under well-watered conditions and is much higher in d1 mutants than in wild-type plants under drought conditions. -
FIG. 10 shows that in the absence of drought stress, increased planting density reduces photosynthesis at light saturation to a greater extent in wild-type than in the d1 mutant. Plants were grown in monospecific stands at the indicated planting densities. Net photosynthesis at 1500 μmol m−2s−1 light was measured on the flag leaf of the primary tiller at 60 days after emergence using a Li-Cor 6400 Portable Photosynthesis System. -
FIG. 11 , from the same experiment asFIG. 10 , shows that high planting density compromises plant growth and survival (as indicated by plant height) earlier in wild-type than in d1 plants. - Higher photosynthetic rates and improved survival at increased planting density are favorable agronomic traits.
FIGS. 10 and 11 show that these traits are exhibited to a greater extent by the d1 mutant than by wild-type plants. - Heterotrimeric G proteins have been implicated in a wide range of plant processes. These include responses to hormones, drought, and pathogens, and developmental events such as lateral root formation, hypocotyl elongation, hook opening, leaf expansion, and silique development. Results and concepts emerging from recent phenotypic analyses of G-protein component mutants in Arabidopsis and rice are adding to our understanding of G-protein mechanisms and functions in higher plants.
- Heterotrimeric GTP-binding proteins (G proteins) provide a key mechanism by which a specific signaling cascade is switched on or off to translate an incoming signal into a specific cellular response. In recent years, much has been learned about the diversity of signal transduction through plant G proteins thanks to the identification and mutation of genes in Arabidopsis and rice (Oryza sativa) that encode specific G-protein components. These components include the α, β, and γ subunits of the G protein heterotrimer, possible heptahelical G-protein-coupled receptors (GPCRs), and regulator of G-protein signaling proteins (RGS). Such studies are revealing two crucial concepts. First, some physiological responses are predominantly mediated by Gα, whereas others are predominantly mediated by Gβγ. Second, the particular role of any given G-protein component in plant developmental processes [1,2,3••] and responses to biotic and abiotic stresses [4-7,8••] can differ in a cell-type- or developmental-stage-specific manner. Thus, one mutant can even show opposite phenotypic responses to the same stimulus, depending on the particular cell or tissue under study. To highlight these concepts, in this review, we discuss the latest genetic studies on plant G-protein signaling from an ‘organ’ point-of-view (
FIG. 1 ). The reader may also be interested in reviews on plant heterotrimeric G proteins that have emphasized comparisons with mammalian systems [9-13]. - The G protein itself consists of three different subunits, α, β, and γ (respectively named Gα, G β, and Gγ), which form a heterotrimeric complex in the inactive state. Binding of an agonist (i.e. an activating ligand) to its specific GPCR leads to the conversion of an inactive G protein to its active conformation. The GPCR acts as a guanine nucleotide exchange factor, causing Gα to exchange GDP for GTP. As a result, Gα-GTP separates from the Gβγ dimer and both Gα-GTP and the Gβγ dimer separate from the receptor and can activate downstream effectors. Subsequent to signal propagation, the GTP that is bound to Gα is hydrolyzed to GDP, thereby inactivating Gα and allowing its re-association with the Gβγ dimer to reform the inactive heterotrimeric complex. RGS proteins act as GTPase-activating proteins (GAPs) for Gα, typically attenuating signaling by hastening the return of the G protein to the resting state.
- Candidate genes that encode polypeptides that are similar to mammalian G-protein components have been isolated from several higher plant species (summarized in [11]). In Arabidopsis and rice, Gα is encoded by a single copy gene, designated GPA1 or RGA1, respectively [14,15]. Gβ is likewise encoded by a single-copy gene, designated AGB1 or RGB1, respectively [16,17]. Two Gγ genes were recently isolated from Arabidopsis and rice: AGG1 or RGG1 [18,19] and AGG2 or RGG2 [19,20]. No plant gene has been found that is highly homologous to metazoan GPCRs. However, in Arabidopsis, GCR1 is a likely candidate as a GPCR-encoding gene because it encodes a protein that has some GPCR sequence similarity and a predicted heptahelical structure that is the hallmark of bona fide GPCRs [8,21,22]. Finally, it appears that the Arabidopsis genome contains only one member of the RGS family, RGS1 [23]. Transgenic plants that ectopically and/or conditionally express each of the above-described components, except the Gγ subunit genes, have been described recently (Table 1), and various single, double, and triple mutants have been generated (Table 1).
- On the basis of both modeling [3•] and experimentation, the basic paradigm of mammalian G-protein signaling described above also appears to operate in plants [13]. Gα, Gβ, Gγ, GCR1 and RGS1 can all be found at the plasma membrane of plant cells [1,8,19,22,23,24,25]. In yeast two-hybrid assays and co-immunoprecipitation experiments, Gβ interacts tightly with both Gγ subunits in both Arabidopsis and rice [18,19,20]. In rice, gel-filtration experiments have confirmed that Gβγ dimers associate with Gα. This association is disrupted by a non-hydrolysable form of GTP, GTPγS, which is expected to maintain the activated conformation of Gα [19]. Pandey and Assmann [8] used in planta and in vitro co-immunoprecipitation as well as split-ubiquitin yeast two-hybrid assays to provide the first conclusive evidence that the putative GPCR, GCR1, physically interacts with Gα. RGS1 interacts with both a constitutively active GPA1 (GPA1QL, the GTPase-deficient version of GPA1) and wild-type GPA1, and the carboxy-terminal domain of AtRGS1 has been shown to exert GAP activity on a yeast Gα [23].
- Striking differences also exist, however, between the G-protein components of plants and those of other eukaryotic organisms: the sequence similarity of the relevant genes and proteins is limited, and a much smaller number of genes encode each of the different components in plants than in other eukaryotes [13].
- Seed germination is a complex phenomenon that is modulated by numerous signals, including gibberellins (GA), abscisic acid (ABA), brassinosteroids (BR), ethylene, light, and sugars, some acting in concert and others in opposition [26]. Current models of seed germination in non-graminaceous species suggest that BR act downstream of GA to promote germination. Both ABA and sugars inhibit germination, and ethylene negatively regulates ABA's effects.
- In the absence of stratification, gpa1-1 and gpa1-2 mutant seeds exhibit delayed germination [27], suggesting that they are more dormant than wild-type seeds. Consistent with this phenomenon, gpa1 mutants exhibit moderately increased sensitivity to the inhibition of germination by ABA and sugars [2,27]. Many of these phenotypes are also observed in Arabidopsis T-DNA insertional mutants of Pirin1, a cupin-domain protein that has been identified as a GPA1 interactor in yeast two-hybrid assays [27].
- Because gpa1 seeds have wild-type ABA concentrations [2], the results described above presumably reflect either an increased sensitivity to ABA or a decreased sensitivity to stimulatory signals such as GA. In support of the latter hypothesis, the germination of gpa1 and agb1 seeds is significantly less sensitive to exogenous GA and significantly more sensitive to the GA-synthesis inhibitor paclobutrazol than the germination of wild-type seeds [2,22]. Ullah et al. [2] speculate that GPA1 controls the sensitivity of the GA pathway because although the overexpression of GPA1 in Arabidopsis confers a millionfold increase in the GA sensitivity of seed germination, the requirement for GA is not abolished. If GPA1 directly coupled the GA response, then the ectopic expression of GPA1 would be expected to confer GA independence, which is not the case. Ullah et al. [2] further suggest that BR controls GA sensitivity in a GPA1-dependent manner, because brassinolide (BL) rescue of germination in seeds treated with paclobutrazol is complete for wild-type seeds but only partial for gpa1 and agb1 seeds [2,22,28].
- Like gpa1 mutants, gcr1 mutants exhibit reduced sensitivity toward GA and BR in seed germination, whereas GCR1 overexpression reduces seed dormancy [22,29]. Under some but not all conditions, seeds of gcr1 gpa1 and gcr1 agb1 double mutants have additive or synergistic germination responses to GA and BR, which is unexpected if GCR1 were to function upstream of the G protein. Thus, under some conditions, GCR1 appears able to act independently of the heterotrimer in regulating seed germination [22].
- Seeds of the rice dwarf1 (d1) mutant, a null mutant [19′] of the rice Gα subunit, RGA1, exhibit a morphological phenotype consisting of short, round grains [30,31]. Observations in rice are also consistent with a role for G proteins in GA-based signaling pathways and the control of transcription in the seed. d1 mutants exhibit reduced GA induction of a-amylase gene expression and enzyme activity in their aleurone cells and reduced expression of the GA-induced genes OsGAMYB and GACa2+ ATPase [32]. Gα may also be a component of BR signaling in rice because BL-stimulated expression of a novel BL-enhanced gene is weaker in d1 mutants than in wild-type seedlings [33]. Ueguchi-Tanaka et al. [32] suggest that there may be two separate GA-signaling pathways in rice, with either high or low sensitivity to GA, and that RGA1 may mediate the former pathway. Both this model and the ‘GPA1 modulation’ model described above [2,32] are consistent with the current data from both Arabidopsis and rice. Hence, additional experimentation, including determination of the ABA sensitivity of d1 seeds, will be required to distinguish between these two possibilities.
- Root growth and architecture involves a balance between cell production in the apical and lateral root meristems and the subsequent elongation of those cells. One advantage of the root as a model system for development is that it is possible to measure rates of cell production and elongation as well as the number of lateral root primordial quite precisely, thereby making it possible to quantify exactly what has changed in the roots of loss- and gain-of function G-protein mutants. The formation of lateral root meristems originates from a set of founder cells that differs from that used to form the primary meristem [34,35]. Therefore, it would not be surprising if the molecular mechanisms that underlie the initiation of lateral and primary root meristems were different. Studies on root meristem formation and maintenance using G-protein mutants are beginning to reveal these mechanistic differences.
- The primary root growth of wild-type plants and that of gpa1 and gcr1 single mutant seedlings appears to be identical in the absence of exogenous hormone treatment [3••,8••]. By contrast, rgs1 mutants have longer primary roots because of their increased cell production rate in the primary root meristem [23••]. rgs1 cells, which lack GAP activity, are predicted to have a greater steady-state pool of activated Gα. This prediction is consistent with the observation that the expression of a transgene that encodes GPA1QL also causes accelerated cell production by the primary root meristem [23••]. This suggests that Gα plays a role in modulating cell division in the primary root meristem. The lack of a large effect of the gpa1 null mutation on primary root growth suggests that the type of modulation that GPA1 exerts may be an increase over a basal state, a state that does not require Ga.
- In response to exogenous treatment with plant growth regulators such as ABA and auxin, primary root elongation is retarded and/or the direction of primary root growth changes. The primary root elongation of gpa1, agb1, or gcr1 single mutants, as well as of double and triple combinations of these mutants, is more sensitive to inhibition by ABA than that of wild-type plants ([8••]; S Pandey, S M Assmann, unpublished). However, the auxin inhibition of primary root length in gpa1 and agb1 mutants is the same as that in wild-type plants [3••], indicating that the dependency of growth inhibition on G proteins differs depending on the hormonal stimulus.
- While the growth of the primary root of gpa1 mutants is like that of wild-type plants under many conditions, the number of lateral roots is greatly increased in agb1 mutants and is decreased in gpa1 mutants [3••]. Opposite to its inhibitory effect on the primary root, auxin is a key activator of lateral root initiation [36]. In the presence of auxin, agb1 plants form more lateral roots, whereas gpa1 plants form fewer lateral roots, compared to wild-type plants [3••]. As is expected if the auxin-induced phenotype for the proliferation of lateral roots is AGB1-dependent, ectopic expression of GPA1 (which is expected to sequester AGB1 into the heterotrimeric complex) also yields an agb1-like phenotype. The expression of GPA1QL has no effect on this phenotype, a finding that is inconsistent with GPA1 acting as a positive modulator of cell division in the lateral root meristem. Thus, Ullah et al. [3••] propose that free Gβγ directly attenuates auxin-induced cell division in lateral roots, as opposed to Gα acting to stimulate this process.
- As for seed germination and root development, several differences have been observed between G-protein-component mutants and wild-type plants during the development of above-ground organs in seedlings and mature plants [10-12].
- When grown in darkness, gpa1 and agb1 seedlings have shorter hypocotyls than wild-type plants because of a reduction in cell number, and these seedlings exhibit partially opened hooks [1,3••,37]. These phenotypes were also observed in gcr1 gpa1 double, agb1 gcr1 double, and agb1 gpa1 gcr1 triple mutants [22]. By contrast, rgs1 mutant seedlings have a longer etiolated hypocotyl as a result of increased cell elongation. This mutant phenotype is similar to that observed in plants that express GPA1QL, consistent with the premise that, in plants as in animals, RGS proteins oppose Gα activation [22].
- When grown in light, gpa1 and agb1 mutants have rounded rosette leaves [1]. The round-leaf phenotype is also found in Arabidopsis gcr1 gpa1 and agb1 gcr1 double mutants and agb1 gcr1 gpa1 triple mutants [22]. Because gcr1 single mutants have wild-type phenotypes for both hypocotyl and rosette-leaf development, GCR1 may not act as the GPCR that is responsible for control of these developmental pathways [22].
- The rice Gα (d1) mutants also exhibit an altered shoot morphology, consisting of broad, dark green leaves and compact panicles [5,30,31,32,38]. One notable contrast between Arabidopsis and rice Gα mutants, however, is that the rice mutants are dwarf but the Arabidopsis mutants are not. Dwarf phenotypes are often associated with GA insensitivity, and GA induction of internode elongation is significantly reduced in the d1 mutants [31]. However, the GA responsiveness of the elongation of the second leaf sheath is similar in d1 mutants and wild-type plants [32]. This selective impairment of GA signaling in d1 mutants suggests cell specificity in GA response, with some pathways being only marginally dependent on Ga.
- During the reproductive phase of plant development, agb1-1 mutants have a floral phenotype consisting of shorter flowers and thicker siliques, but this phenotype is not shared by gpa1 mutants [1,3••,39]. Constitutive overexpression of GPA1 reduces silique length, producing a phenotype that is similar to that of agb1. This evidence is consistent with the idea that silique length is controlled by released Gbg [3••]. gpa1 sepals and pedicels are longer, whereas agb1 sepals are shorter, than those of wild-type plants [3••], findings that are again consistent with a Gβγ-dependent pathway. Transformants that overexpress GCR1 flower earlier [29], but gcr1 null mutants typically do not flower later, than wild-type plants [22].
- G proteins are implicated in several stress-signaling pathways in plants. In mature leaves, G proteins transmit signals to molecules, including small GTPases, ion channels, and phospholipases, that are effectors in the responses to various biotic and abiotic stress conditions, including pathogen elicitation, ozone treatment and water deficit.
- There are no reports as yet on pathogen signaling in Arabidopsis G-protein mutants, but some defense signaling pathways in rice appear to rely on RGA1. Upon infection with a virulent strain of bacterial blight (Xanthomonas oryzae pv. Oryzae [Xoo]), symptom development in d1 mutants is more severe than that in wild-type plants [7]. By contrast, infection with virulent strains of rice blast fungus (Magnaporthe grisea) produces identical lesions in d1 mutants and wild-type plants. d1 mutants exhibit a highly reduced response, however, upon inoculation with avirulent rice blast [5,7]. Expression of a constitutively active OsRac1 in d1 mutants restores defense signaling and resistance, suggesting that RGA1 functions upstream of this small GTPase [5]. Yet, in a d1 mutant cell line treated with the oligosaccharide elicitor chitin, the elicitation of defense responses such as extracellular alkalinization, generation of reactive oxygen species, phytoalexin accumulation and the induction of specific genes does not differ from that of wild-type cells [40,41]. Taken together, these studies indicate that the extent of G-protein coupling of responses to both avirulent and virulent pathogens is pathogen- and elicitor-specific.
- Like pathogen infection, exposure to high ozone (O3) levels results in foliar lesions, and O3 responses share signaling pathways and gene expression patterns with the hypersensitive response [42]. gpa1 null mutants and the double mutant gpa1-4 agb1-2 respond differently to O3 compared to wild-type plants, and to gcr1 and rgs1 single mutants. The major difference observed among these mutant genotypes is an O3-resistant phenotype of the gpa1 lines, indicated by lack of leaf curling in response to O3 [6].
- One of the phenomena commonly observed following O3 exposure is a reduction in stomatal apertures [43], a response that is also evoked by ABA. gpa1 mutants exhibit reduced O3 sensitivity at the whole-leaf level. At the single (guard)-cell level, gpa1 mutants also exhibit aspects of ABA insensitivity, including reduced ABA inhibition of guard cell inward K+ channels and altered ABA-promotion of slow anion currents [4]. Recently, the lipid metabolite, sphingosine-1-phosphate (SIP), has been described as a secondary messenger for ABA responses [44,45••]. The guard cells of gpa1 mutants show insensitivity to inhibition of stomatal opening by either ABA or SIP. However, ABA still induces wild-type levels of stomatal closure in gpa1 [4], whereas stomatal closure in this genotype is insensitive to SIP. This difference implies that the SW response is obligatorily mediated by GPA1, whereas there is a parallel or backup pathway for ABA induction of stomatal closure that is independent of GPA1 [45••].
- gcr1 mutant guard cells exhibit hypersensitivity to ABA and SP in both inhibition of stomatal opening and promotion of stomatal closure [8••], which would be unexpected if GCR1 were to transmit the ABA signal to GPA1. Pandey and Assmann [8••] therefore proposed that GCR1 acts as a negative regulator of GPA1-mediated ABA responses in guard cells. Consistent with this phenomenon, gcr1 mutant plants have higher expression levels of some known drought- and ABA-regulated genes after exogenous ABA treatment and exhibit improved recovery following drought stress [8••].
- Many enzymes, including phosphatidylinositol-phospholipase Cs (PLCs; reviewed in [46]) and phospholipase Ds (PLDs; [47,48]), act as effectors of the ABA response during the regulation of stomatal aperture. These phospholipases also have been identified recently, albeit not yet in guard cells, as intracellular effectors of G protein signaling. For instance, using tobacco BY2 cells that overexpressed GCR1, Apone et al. [49] concluded that GCR1 regulates DNA synthesis through activation of PLC. In Arabidopsis, PLDα1 directly binds GPA1 via a motif similar to the DRY motif that is present in many mammalian GPCRs [50••]. Binding inhibits PLDα1 activity and is relieved upon GTP addition, suggesting that, in vivo, G protein activation leads to the activation of PLDa1 [50••]. Thus, it will be of interest to assess PLC and PLD activity in guard cells in which the levels of G-protein components are altered.
- As is evident from the phenotypes described in this review, numerous processes at all stages of plant development are modulated by heterotrimeric G proteins. Many of these phenotypes appear upon null mutation of the Gα subunit genes GPA1 or RGA1, implying dependency upon Gα coupling to downstream effectors. However, some phenotypes, notably lateral root proliferation and altered silique morphology, are present in agb1 mutants but are either absent or opposite in gpa1 mutants, implying a dependency on Gβγ-coupled signaling. But is that the whole story? Plausibly, the different phenotypes of gpa1 and agb1 mutants could reflect differences in the relative levels of the released subunits from the heterotrimeric complex, different fluxes of signaling through Gα (as opposed to Gβγ) in the different cell types or organs, and/or a different relative balance in positive or negative feedback. To sort out these issues, it will be necessary to determine the effect on a given trait of quantitatively altered ratios of Gα to Gβγ, rather than of the two extremes of ratios of 0 or ∞ that are created by single null mutations. Given the plethora of G-protein-related phenotypes in combination with the dearth of heterotrimeric G-protein subunits in plant genomes, one might well predict that plants will be found to have evolved novel and abundant mechanisms for coupling G protein components with downstream effector molecules.
- In Arabidopsis, seeds and light-grown gpa1 seedlings show increased sensitivities to ABA and sucrose together with decreased sensitivities to BL [2,27′], suggesting that identical G-protein-based signaling pathways may operate in seed germination and early seedling development. In mature rosette leaves, however, gpa1 guard cells exhibit reduced rather than enhanced sensitivity to ABA. In rice, internode sensitivity to GA is strongly reduced in d1 mutants, yet GA-regulation of leaf-sheath elongation is scarcely affected. These differential sensitivities indicate that the roles of GPA1 must be cell- and tissue-specific, presumably reflecting cell- and tissue-specific effectors and/or GPCRs. Cell-specific mechanisms for G-protein-coupled signaling have precedent in animal systems (e.g. [51]).
- By parallel reasoning, one might expect a proliferation of cell-specific GPCRs in plants. However, if this is true, the plant GPCRs must be defined by functionality rather than by sequence similarity; GCR1 is the sole candidate GPCR to be identified in Arabidopsis on the basis of homology criteria and its sequence similarity to known GPCRs is limited. The observations that GCR1 is not implicated in many of the pathways that are affected by mutation of GPA1 and/or AGB1, and that the ABA-related phenotypes of gcr1 mutants are opposite to those of gpa1 mutants, further highlight our lack of knowledge about components that function upstream of plant G-protein heterotrimers. Signals may be transduced either via novel GPCRs or through proteins that transmit signals to G proteins independently of GPCRs [51,52]. Furthermore, plant-specific ‘unconventional’ G proteins, such as A. thaliana Extra Large G Protein1 (XLG1), a protein that has significant similarity to Gα subunits and exhibits GTP-binding capability [11,53], could potentially partner with components of G-protein pathways. Thus, the future is bright for model plant systems such as Arabidopsis and rice to contribute new insights regarding this ubiquitous eukaryotic signaling paradigm.
- In Arabidopsis, GCR1 may positively regulate seed germination by coupling BR promotion of GA-stimulated germination. GCR1 also can act independently of GPA1 and AGB1 in a pathway to regulate GA-stimulated germination [23••]. RGS1 antagonizes the activation of GPA1 [23••]. Pirin1 may positively regulate seed germination by overcoming the negative effect of ABA or by activating germination-promoting pathways [27′]. In rice, RGA1 may work in a high-sensitivity GA pathway that regulates the induction of Ca2+-ATPase and α-amylase, leading to seed germination [32]. RGA1 may also be a component of BR signaling [33]. In addition, there may be an alternative GA pathway that also induces a-amylase but does not involve RGA1 [32]. (b) Cell division/elongation. During seedling growth, GPA1, AGB1 and GCR1 may act in the inhibition of primary root development by ABA ([8••]; S Pandey, S M Assmann, unpublished). Furthermore, GCR1 negatively regulates ABA-induced gene expression [8••]. AGB1 and GPA1 activate cell division in both hypocotyls and leaves [1,2,3••,37] whereas RGS1 antagonizes the activation of GPA1 in apical root meristems [23••]. Auxin treatment also increases GPA1 transcript levels and decreases AGB1 transcript levels (not shown in figure, [3••]). During lateral root formation, AGB1 functions downstream of GPA1 and inhibits auxin-induced cell division, and GPA1 inhibits AGB1 function [3••]. (c) Stress responses. According to the leaf curling phenotype, GPA1 promotes the 03 sensitivity of Arabidopsis plants [6]. Drought stress and ABA treatment inhibit stomatal opening and promote stomatal closure. ABA triggers SP formation, which is coupled by GPA1 to inhibit plasma membrane inwardly rectifying K+ channels and to activate slow anion channels, resulting in the inhibition of stomatal opening and the promotion of stomatal closure [4,45••]. The GPCR-like protein GCR1 directly binds to GPA1 and negatively controls ABA- and S1P-regulation of stomatal apertures [8]. In rice, responses to avirulent rice blast fungus, including the accumulation of transcripts for the small GTPase, OsRac1, are attenuated in the RGA1 mutant d1. OsRac1 acts as a key molecular switch for multiple signaling pathways, such as the production of reactive oxygen species that lead to disease resistance. Expression of constitutively active OsRac1 in the d1 mutant restores defense signaling [5]. In response to virulent strains of bacterial blight, lesions are more severe in the d1 mutant than in wildtype plants [7]. (d) Morphology. In Arabidopsis, both GPA1 and AGB1 modulate leaf development and shape [1,39]. gpa1 and agb1 mutants exhibit rounded lamina. AGB1 is also involved in flower and fruit development [1,3••,39]. In agb1, the floral buds at the inflorescence apex are more tightly clustered, the siliques are shorter, and the silique tips are more blunt than those of wildtype plants. In rice, RGA1 modulates plant stature by regulating internode and panicle elongation, and also influences the color of leaf blades and sheaths and grain shape [32].
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TABLE 1 Mutant and transgenic lines for Gα, Gβ, GCR1 and RGS 1Name/Allele Ecotype/cultivar cDNA Status of transcript/translation product Phenotype comparison with wild-type ecotypes Mutant loss of function for Gα subunit in Arabidopsis (A. thaliana) Phenotypes of gpa1 mutants gpa1-1 Ws T-DNA insertion in 7th intron. Lacks full-length transcript Less sensitive to GA and BL stimulation of germination Wisconsin KO Arabidopsis facility α population [1]. [1, 2]. More sensitive to the GA biosynthesis inhibitor paclobutrazol [2, 22]. Hypersensitive to ABA and sugar inhibition of germination [2, 27*]. In darkness, partial deetiolation: open hook; shorter hypocotyls caused by reduced cell division [1]. gpa1-2 Ws T-DNA insertion in 8th exon. Lacks full-length transcript Primary root forms fewer lateral root primordia [1]. Wisconsin KO Arabidopsis facility [1]. Less sensitive to auxin promotion of laterial root formation α population. [3**]. Rounded lamina shape [1, 37]. Leaf cells are fewer and larger [1]. Longer sepals and pedicels [1]. Less sensitive to O3 [6]. gpa1-3 Col-0 T-DNA insertion in 9th exon. Lacks full-length transcript More water loss [4]. Salk collection. [37]. Insensitive to ABA inhibition of stomatal opening [4]. Insensitive to ABA inhibition of inward K+ channels [4]. Insensitive to S1P promotion of stomatal closure [45**]. gpa1-4 Col-0 T-DNA insertion in 12th itron. Lacks full-length transcript Altered sensitivity to ABA activation of slow anion channels Salk collection [37]. [4]. Insensitive to S1P activation of slow anion channles [45**]. Phenotypes of Daiokoku dwarf1 (d1) mutants (DK22, Mutant loss of function for Gα subunit in rice (Oryza sativa) HO541, CM 1361-1, T65d1, rga1) DK 22 Nipponbare Point mutation of G598 to T in 8th Stop codon generated Shorter and rounded grains [30, 31]. exon. [31, 40]. Reduced GA and BL stiumlation of gene expression [32, 33]. Protein null [19*]. Ho 541 Nipponbare Spontaneous mutant: Deletion of RGA1 transcript null [3-] Shorter and darker green leaves, more compact particle [5, 30-32, 833 basepairs between 1st exon and 38]. intron. CM 1361-1 Kinmaze Insertion between nucleotides 354-355 Predicted protein lacks Shorter internodes - may be due to a decrease in the number GTP-, effector- and of cells per internode [31]. receptor-binding regions Reduced GA stimulation of internode growth [32]. [31]. Normal GA stimulation of second leaf sheath elongation [32]. T65d1 Taichung 65 Deletion of nucleotides 1003-1004. Stop condon generated Reduced hypersensitive response to infection by rice blast before third effector-binding fungus [5]. region [32]. rga1 Nipponbare Antisense suppression. RGA1 transcript null line Increased sensitivity to infection by virulent strain of [31] bacterial blight [7]. HO 532 HO 533 HO 537 Nipponbare Spontaneous mutants. [30] Not used for phenotypic analysis. HO 538 HO 552 FL2 Nipponbare Marker line derived from HO 538. [30] Not used for phenotypic analysis. ID 1Shiokari Deletion of nucleotides 1003-1004. [31] Not used for phenotypic analysis. CM392; Kinmaze Induced by N-methyl- [5, 30] Not used for phenotypic analysis. 1729; 248; N-nitrosourea. 723; 1232 DKT 1Taichung 65 Point mutation of A1075 to T. [31] Not used for phenotypic analysis. DK 2Taichung 65 Deletion betwween nucleotides Predicted to lack GTP- Not used for phenotypic analysis. 932-979 binding region [31]. Mutant gain of function for Gα subunit in Arabidopsis Phenotypes of GPA1 overexpressors Q222L or Col 35S promoter::point mutation Mutation disables GTPase No effect on auxin-induced cell divison in lateral roots [3**]. GPA1* A1264 to T derived from GPA1 activity, leading to No auxin effect on hypocotyl length [3**]. cDNA. constitutively active Gα [1, 3**, 37]. GPA1α Ws 35S promoter::constitutive form of Overexpression of Increased hypocotyl length caused by increased cell GPA1 cDNA constitutively active Gα elongation [23**]. (GPA1QL) [23**]. Longer primary roots caused by increased cell production [23**]. cGα Ws DEX inducible Overexpression of Under low light condition, shorter hypocotyls are caused by a promoter::constitutive form of constitutively active Gα reduction of cell elongation, also smaller cotyledons and GPA1 cDNA. (GPA1QL) [54]. increased stomatal density in hypocotyl [54]. wGα Ws DEX inducible promoter:: Overexpression of full- Under low light condition, shorter hypocotyls are caused by a GPA1 cDNA. length GPA1 protein [54]. reduction of cell elongation, also smaller cotyledons and increased stomatal density in hypocotyl [54]. gpa1 Col-0 DEX inducible promoter:: Complementation in gpa1 Hypersensitive to GA stimulation of germination [2]. (GPA1) GPA1 cDNA. background [3**, 37]. In darkness, shorter hypocotyls [37]. Hypocotyl hypersensitive to auxin-induced adventitious root formation [3**]. 35S::GPA1- Col 35S promoter::GPA1 cDNA fused Overexpression of GFP with GFP. fluorescent GPA1 [23**]. GOX Col DEX inducible promoter: Overexpression of GPA1 Mimics agb1-2 lateral root phenotype: more lateral roots GPA1 cDNA: [2, 37]. [3**]. GOX1 Tobacco cells DEX inducible promoter:: Transformed BY2 cells Shorter cell cycle [1]. Nicotiana GPA1 cDNA. overexpressing GPA1 Higher Ptdlns-PLC activity [49]. tabaccum cv. [1, 49]. Higher Ins(1, 4, 5)P3 content [49]. BY2 Mutant gain of function for Gα subunit in rice Phenotypes of RGA1 overexpressors QL/d1 or Nipponbare 35S promoter:: point mutation Expression of constitutively Active form GTP-Gα presents fee from Gβ or Gγ subunits Q223L Q223 to L derived from RGA 1active Gα in d1 background [9*]. cDNA. [19*]. Mutant loss of function for Gβ subunit in Arabidopsis Phenotypes of agb1 mutants agb1-1 or Col Ethyl methanesulfonic acid Mutant transcript slightly Less sensitive to GA and BR stimulation of germination [22]. elk4 mutagerized. Missense mutation: larger because of splicing More sensitive to the GA biosynthesis inhibitor paclobutrazol failure to splice out the 1st intron. failure. Stop condon [2, 22]. Hypersensitive to sugar inhibition of germination [2]. generated [39]. In darkness, partial de-etiolation: open hook; shorter hypocotyls caused by reduced cell division [1]. Hypocotyl hypersensitive to auxin-induced adventitious root formation [3**]. Primary root forms more lateral root primordia [3**]. agb1-2 Col-0 T-DNA insertion in 4th exon. Lacks full-length transcript More sensitive to auxin promotoino of lateral root formation Salk collection. [3**]. [3**]. Rounded lamina shape and presence of islands of small cells that create a crinkly surface [3**. 39]. Shorter flowers and sepals [39]. Shorter and thicker siliques [39]. Mutant gain of function for Gβ subunit in Arabidopsis Phenotypes of AGB1 agb1-1 Col Transformation with genomic Complementation in agb1-1 (AGB1) fragment containing AGB1 gene background [39]. and promoter. agb1-1 Col-0 DEX inducible promoter:: Complementation in agb1-2 Decreased auxin-induced lateral root formation relative to (AGB1) AGB1 cDNA. background [3**]. agb1 [3**]. BOX Col-0 DEX inducible promoter:: Overexpression of AGB1 AGB1 cDNA. [3**, 37]. Mutant loss of function for GCR 1 in ArabidopsisPhenotypes of gcrf mutants gcr1-1 Col-0 T-DNA insertion in 8th intron. Lacks full-length transcript Less sensitive to GA and BR stimulation of germination [22]. Salk collection. [22]. Hypersensitive to ABA inhibition of germination [8**]. gcr1-2 Col-0 T-DNA insertion in 6th exon. Lacks full-length transcript Hypersensitive to the GA inhibitor paclobutrazol [22]. Salk collection. [22]. Increased ABA promotion of ABA-regulated gene expression [8**]. gcr1-3 Ws T-DNA insertion in 2nd intron. Lacks full-length Increased resistance to drought stress [8**]. Wisconsin Arabidopsis KO transcript[8**]. Hypersensitive to ABA and S1P inhibition of stomatal facility BASTA population. opening [8**]. gcr1-4 Col T-DNA insertion in 3rd intron. Lacks full-length transcript Hypersensitive to ABA and S1P promotion of stomatal SAIL collection of TMRI. [8**]. closure [8**]. Flowers slightly earlier [22]. Mutant gain of function for GCR1 in Arabidopsis Phenotypes of GCR1 overexpressors gcr1-3 Ws DEX inducible promoter::GCR1 Expression in gcr1-3 GCR1-FLAG immunoprecipitates with GPA1 [8**]. (GCR1) cDNA fused with FLAG tag. background [8**]. 35S::GCR1- Col 35S promoter::GCR1 Overexpression of GFP cDNA fused with GFP. fluorescent GCR1 [22] GCR1-over- Col 35S promoter::GCR1 Overexpression of GCR1 Lacks seed domancy [29]. expressing cDNA [29]. Increawsed expression of germination associated genes [29]. lines Early flowering [29] GCR1-over- Tobacco cells 5S promoter::GCR1 Transformed BY2 cells Increased DNA synthesis [29]. expressing Nicotiana cDNA overexpressing GCR1 Higher PtdIns-PLC activity [49]. BY2 cells tabaccum cv. [29, 49]. Higher Ins(1, 3, 5)P3 content [49]. BY2. Mutant loss of function for RGS1 in Arabidopsis Phenotypes of rgs1 mutants rgs1-1 Col-0 T-DNA insertion in 6th intron. Lacks full-length transcript Mimics GPA1QL phenotype under darkness: longer Salk collection. [23**]. hypocotyls caused by increased cell elongation [23**]. Longer primary roots caused by increased cell production in light [23**]. rgs1-2 Col-0 T-DNA insertion in 9th intron. Lacks full-length transcript Insensitive to 6% D-glucose inhibition of seedling growth Salk collection. [23**]. [23**]. Mutant gain of function for RGS1 in Arabidopsis Phenotypes of RGS1 overexpressors 35S::RGS1- Col 5S promoter::RGS1 Overexpression of GFP cDNA fused with GFP fluorescent RGS1 [23**]. ROX Col-0 DEX inducible promoter:: Overexpression of full Mimics gpa1 mutant hypocotyl phenotype under darkness: RGS1 open reading frame length RGS1 protein [23**]. shorter hypocotyl [23**]. Hypersensitive to 6% D-glucose inhibition of seedling growth [23**]. Loss of function double/triple mutants in Arabidopsis Phenotypes double/triple mutants gpa1-4 Col-0 Cross between gpa1-4 and agb1-2. [37] Less sensitive to GA and BR stimulation of germination agb1-2 (same sensitivity as agb1 mutant [22]). Shorter hypocotyls and partially opened hooks [37]. Rounded lamina shape [22]. Less sensitive to O3 [6]. gcr1-2 Col-0 Cross between gcr1-2 and gpa1-4 [22, 37] Less sensitive to GA and BR stimulation of germination gpa1-4 agb1-2. (additive or synergistic effect of mutations [22]). gpa1phenotype under darkness: shorter hypocotyl and partially opened hook [22]. gpa1 leaf morphology: rounded lamina shape [22]. Less sensitive to GA and BR stimulation of germination (additive or synergistic effect of mutations [22]. agb1-2 Col-0 Cross between gpa1-4 agb1-2 and [22] agb1 phenotype under darkness: shorter hypocotyl and gcr1-2 gcr1-2. partially opened hooks [22]. agb1 leaf morphology: rounded lamina shape [22]. agb1-2 Col-0 Cross between gpa1-4 [22] Less sensitive to GA and BR stimulation of germination gcr1-2 agb1-2 and gcr1-2. (additive or synergistic effect of mutations [22]). gpa1-4 gpa1 and agb1 phenotype under darkness: shorter hypocotyl and partially opened hooks [22]. gpa1 and agb1 phenotype under darkness: shorter hypocotyl and partially opened hooks [22]. gpa1 and agb1 leaf morphology: rounded lamina shape [22]. agb1-1 Col Cross between agb1-1 and [39] Shorter petiole, shorter lamina than either agb1 or er105 er-105 receptor-like kinase erecta mutant, single mutant, suggesting that ER and AGB1 function in er-105 parallel pathways controlling these characteristics [39]. D1 slr Nipponbare Cross between d1 and GA [32] SLR is epistatic to D1 supporting RGA1 involvement in GA insensitive mutant slr. signaling [32]. 35S, cauliflower mosaic virus 35S promoter; BOX, AGB1 overexpressing lines; Col, Columbia; DEX, dexamethansone; elk4, erecta like 4 mutant; er-105, receptor-like kinase erecta mutant; GFP, green fluorescent protein; GOX, GPA1 overexpressing lines; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; PtdIns-PLC, phosphatidylinositol-phospholipase C; ROX, RGS1 overexpressing lines; SAIL, Syngenta Arabidopsis Insertion Library; slr, slender rice mutant; TMRI, Torrey Mesa Research Institute; Ws, Wassilewskija. -
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- Phenotypic plasticity is the ability of one genotype to display different phenotypes under different environmental conditions. Although variation for phenotypic plasticity has been document in numerous species, little is known about the genetic mechanisms underlying phenotypic plasticity. Given their widespread roles in hormonal and environmental signaling, we examined whether genes which encode heterotrimeric G proteins are plasticity genes.
- We grew multiple alleles of heterotrimeric G-protein mutants, together with wild-type Arabidopsis thaliana, under different watering regimes to determine the contributions of G-protein genes to phenotypic plasticity for a number of developmental and reproduction-related traits.
- G-protein mutations did not affect significantly the amount of phenotypic variation within an environment for any trait, but did affect significantly the amount of phenotypic plasticity for certain traits.
- AGB1, which encodes the 13 subunit of the heterotrimeric G protein in Arabidopsis, is a plasticity gene and regulates reproductive trait plasticity in response to water availability, resulting in increased fitness (defined as seed production) under drought stress.
- Heterotrimeric G proteins are multisubunit guanosine triphosphate (GTP)-binding proteins that function in the transduction of external signals into cellular responses. Because G proteins regulate a large array of cellular and developmental processes in both plants and animals, it is of interest to evaluate their potential impact on developmental plasticity and fitness. According to the paradigm of G-protein signaling, the G protein is activated following the binding of a ligand to an associated membrane-bound G-protein-coupled receptor (GPCR). This binding results in a conformational change in the alpha subunit (Gα) and the subsequent exchange of GTP for guanosine diphosphate by Gα, resulting in the dissociation of Gα from the beta gamma dimer (Gβγ). Gα and/or Gβγ are then free to interact with downstream signal effectors until the intrinsic GTPase activity of Gα results in the reassembly of the inactive trimer (Assmann, 2002).
- In mammals, there are a number of genes which encode heterotrimeric G-protein subunits and hundreds of GPCRs have been predicted, resulting in a large, diverse assortment of potential G-protein signaling pathways (Fredriksson & Schioth, 2005; McCudden et al., 2005). Numerous ligands have also been identified for mammalian GPCRs, including light, sensory molecules including odors and tastes, hormones, neurotransmitters and bacterial toxins (Civelli, 2005), and mutations in mammalian G-protein subunits often result in genetic disorders (Spiegel & Weinstein, 2004; Weinstein et al., 2006).
- Plants possess few genes which encode G-protein subunits, and mutations in these genes, despite their broad expression throughout the plant body, do not result in lethality or in extreme phenotypes under ‘ideal’ laboratory growth conditions (Perfus-Barbeoch et al., 2004). The Arabidopsis thaliana genome contains single genes encoding Gα (GPA1) (Ma et al., 1990) and Gb (AGB1) (Weiss et al., 1994) subunits, and two known Gγ (AGG1 and AGG2) (Mason & Botella, 2000, 2001) genes. GPCR diversity is also reduced in Arabidopsis; only one putative GPCR, GCR1, has been functionally characterized (Pandey & Assmann, 2004), although several dozen additional genes have been predicted to function as GPCRs based on topology prediction (Moriyama et al., 2006; Gookin et al., 2008) and coupling to GPA1 in yeast (Gookin et al., 2008). To date, some phenotypes of Arabidopsis G-protein mutants, such as leaf morphology and lateral root production (Ullah et al., 2001, 2003), have been shown to be regulated by G-protein subunits in congruence with classical mammalian paradigms, whereas other phenotypes, such as root waving and control of floral and fruit morphology (Pandey et al., 2008; Trusov et al., 2008), suggest unique variants of plant G-protein regulatory modes (Assmann, 2005; Temple & Jones, 2007). Such variants may be mechanistically related to plant-specific aspects of the G-protein complement, including the slow GTPase activity of the plant Gα subunit (Johnston et al., 2007; Pandey et al., 2009), the existence of an unusual class of plant-specific Gα-like proteins, the extra-large G proteins (Lee & Assmann, 1999; Pandey et al., 2008) and the presence in Arabidopsis of novel GPCR-like regulatory proteins, the GTG and RGS proteins (Chen et al., 2003; Pandey et al., 2009).
- Despite the paucity of heterotrimeric G-protein subunits in the Arabidopsis genome, functional studies of G-protein mutants have shown diverse roles for heterotrimeric G proteins in germination, development, phytohormone responses [abscisic acid (ABA), auxin, brassinosteroids, gibberellins], stress responses (ozone, reactive oxygen species, pathogens) and stomatal aperture regulation (Ullah et al., 2001, 2002, 2003; Wang et al., 2001, 2007; Pandey & Assmann, 2004; Joo et al., 2005; Llorente et al., 2005; Pandey et al., 2006; Fan et al., 2008; Zhang et al., 2008a; Zhang et al., 2008b). How a limited number of heterotrimeric G-protein subunits can transduce such a large number of hormonal and environmental signals is a fundamental question in plant G-protein signaling (Assmann, 2004). Further characterization of GTGs and other unconventional G proteins, such as the XLGs (Lee & Assmann, 1999; Ding et al., 2008), and the identification of GPCR ligands, potential tissue-specific GPCRs, and G-protein signaling effectors in plants may help to elucidate this question. An additional model which has been proposed is that G proteins may serve as signal modulators instead of direct transducers of signals (
FIG. 1 ). By functioning as ‘cross-talk hubs’, G proteins could fine tune a phenotype or physiological response based on multiple signals/environmental inputs (Assmann, 2004). This model also addresses another paradox in plant G-protein signaling: why, if G proteins are important to plant physiology, are G-protein mutations not lethal in plants? A mutation may not be lethal if additional copies or similar versions of the gene exist in the genome or if the expression of the gene is specific to a certain stress (e.g. cold shock) or to a less vital plant tissue or cell type (e.g. trichomes). However, none of these situations applies to Arabidopsis as Gα and Gβ are both encoded by single, canonical genes which are widely expressed throughout the plant (Ma et al., 1990; Huang et al., 1994; Lease et al., 2001; Anderson & Botella, 2007). Alternatively, if G proteins function in directing hormonal and environmental cross-talk, they may be required only in the production of the ‘optimal’ phenotype, and the direct transducers of the signal would still function in the absence of functional G-protein subunits. - Plants, being sessile organisms, are hypothesized to have evolved increased phenotypic plasticity, the ability of one genotype to display different phenotypes under different environmental conditions, compared with their mobile animal counterparts (Bradshaw, 1972; Schlichting, 1986; Sultan, 1987; Huey et al., 2002). Heightened plasticity in plants would allow plants to compensate for inescapable and inhospitable environments. Although variation for plasticity has been documented in numerous plant and animal species, and many theories have been proposed concerning the ecological and evolutionary significance of this variation, very little is known about the explicit genetic machinery which underlies phenotypic plasticity.
- The phenotypic plasticity (or lack thereof) of a trait can be graphically represented by a reaction norm, which is a plot of the mean phenotypic value of the trait in different environmental conditions (
FIG. 2 ). A horizontal reaction norm indicates that the trait lacks plasticity, whereas a line with a nonzero slope or a curved line is indicative of phenotypic plasticity. As plasticity genes control the shape or slope of the reaction norm of a trait, when these genes are mutated, it is expected that the shape/slope of the reaction norm will diverge from that of the wild-type. Mutations may alter the height of the reaction norm without affecting the overall plasticity for a trait (line C), affect the amount of plasticity for a trait (line B) or may change the direction of plasticity for a trait (line D). Differences in reaction norm shapes (plasticities) among genotypes can be detected and tested using analysis of variance (ANOVA)-based statistical methods. Specifically, a significant genotype×environment/treatment interaction term for a trait indicates that there is variation for plasticity among the genotypes, that is, the response curves have different shapes/slopes. - Although functional analysis of specific genic mutants is a widespread method to determine gene function, this technique has been applied to phenotypic plasticity studies only in a few instances, which have focused mainly on photoreceptor genes. For example, in Arabidopsis, this approach has been employed to study the genetic basis of phenotypic plasticity in photomorphogenetic responses (Pigliucci & Schmitt, 1999, 2004). Arabidopsis hy1 and hy2 photoreception mutants, which display constitutively active shade avoidance responses, showed reduced fitness under some environmental conditions relative to the more plastic wild-type (Pigliucci & Schmitt, 1999). This result suggests that HY1 and HY2 (which encode a plastid heme oxygenase and a phytochromobolin synthase, respectively) (Muramoto et al., 1999; Kohchi et al., 2001) are plasticity genes. In addition, naturally occurring polymorphism of the photochrome PHYB locus has been associated with altered light responses in Arabidopsis; however, phenotypic plasticity was not measured explicitly (Filiault et al., 2008). In field experiments, plants harboring mutations in phototropin blue light photoreceptors showed reduced fitness relative to the wild-type under a range of light conditions; however, significant genetic variation for phenotypic plasticity was observed only for the seedling emergence rate (Galen et al., 2004).
- Genetic variation for plasticity has also been documented among wild-type accessions of Arabidopsis (Pigliucci & Kolodynska, 2002; Schmuths et al., 2006; Brock & Weinig, 2007), and naturally occurring variation for plasticity within plant species has been assessed in quantitative trait locus (QTL)-based phenotypic plasticity studies. Using recombinant inbred lines in lieu of genetic mutants, these studies have lent additional support for the existence of plasticity genes. Studies on recombinant inbred lines of Arabidopsis (Kliebenstein et al., 2002; Ungerer et al., 2003; Juenger et al., 2005), barley (Lacaze et al., 2009) and poplar (Wu, 1998) found significant QTL×environment interaction for a number of traits. Specifically with regard to drought stress in Arabidopsis, Hausmann et al. (2005) found significant recombinant inbred line×environment interactions for a number of water use traits, further supporting the existence of genes which regulate phenotypic plasticity to drought stress in Arabidopsis.
- The signaling cross-talk mechanism discussed above may be an important component of phenotypic plasticity, as phenotypes could be tweaked on the basis of multiple environmental inputs. A mutation in a G-protein subunit might decrease cross-talk and therefore reduce the plant's ability to adjust a phenotype or response to changing environments based on multiple signals. Specifically, a G-protein mutation might affect the degree of plasticity of a trait (
FIG. 2 , line B), the amount of variation within an environment for a trait (that is the ‘noisiness’ of the trait) or might simply shift the mean value of the trait away from the wild-type value (FIG. 2 , line C) (Assmann, 2004). Therefore, by studying populations of G-protein mutants under multiple environments, we might reveal whether G-protein genes are plasticity genes, as well as evaluate the importance of G proteins to plant fitness. In addition, previous studies of heterotrimeric G proteins have, to a large extent, focused on guard cell physiology (Wang et al., 2001; Pandey & Assmann, 2004; Fan et al., 2008) and cell division (Ullah et al., 2001, 2003; Chen et al., 2006). By studying whole-plant phenotypic plasticity responses of G-protein mutants, we might reveal novel functions of G proteins that could not be identified by previous cell-centered approaches. - As G-protein signaling has been implicated in stomatal aperture regulation (Wang et al., 2001; Pandey & Assmann, 2004; Fan et al., 2008), stomatal density (Zhang et al., 2008a) and seed and seedling ABA responses (Pandey et al., 2006), we chose G-protein regulation of phenotypic plasticity and plant fitness under drought as the focus of the present study. The following questions were asked:
- 1. Do G-protein mutations alter the shape/slope of reaction norms in response to water availability, that is are G-protein genes ‘plasticity genes’?
- 2. Does a G-protein mutation affect the level of phenotypic variation within an environment? 3. What are the fitness consequences of G-protein mutations in different environments?
- Using multiple alleles of gpa1, agb1 and gcr1 mutants, we found significant variation for plasticity for a number of reproduction-related traits in response to water availability. agb1 mutants showed significantly reduced plasticity for inflorescence height, number of fruits and seed number per fruit. Interestingly, agb1 mutants showed enhanced fitness under drought stress compared with the wild-type, but all G-protein mutants showed reduced fitness under ample water conditions. These data support the hypothesis that heterotrimeric G-protein genes are indeed plasticity genes in plants.
- Plant Growth Conditions and Water Treatments
- All Arabidopsis thaliana (L.) Heynh seeds used in this experiment were collected from parent plants that were grown together under uniform conditions. gpa1-3, gpa1-4, agb1-1, agb1-2, gcr1-1, gcr1-2 and gpa1-4agb1-2 mutants have all been described previously and were generated using the ecotype Col (Lease et al., 2001; Jones et al., 2003; Ullah et al., 2003; Chen et al., 2004). All mutants are TDNA insertional mutants, with the exception of agb1-1, which is an ethylmethanesulfonate-generated point mutation (Lease et al., 2001). It has been determined that gpa1-3 and gpa1-4 are null mutants at both the transcript and protein levels (Jones et al., 2003). agb1-2, gcr1-1 and gcr1-2 are all transcript null alleles (Ullah et al., 2003; Chen et al., 2004). agb1-1 produces a larger and less abundant AGB1 transcript compared with the wild-type as a result of a destabilizing point mutation, which results in a failure to splice out the first intron and the introduction of a premature stop codon (Lease et al., 2001). All alleles were backcrossed once in our laboratory and the genotypes of parent plants were confirmed via PCR of genomic DNA. Seed storage was identical for all seed lots. Cold stratified seeds (stratified at 4° C. for 48 h in darkness on wet filter paper) were sown directly on the surface of a soil mix composed of Miracle-Gro potting mix (The Scotts Co, Marysville, Ohio, USA), Turface Greens Grade fritted clay (Profile Products LLC, Buffalo Grove, Ill., USA) and perlite in a 16:8:1 volume ratio. The plants were grown in Kord 90 mm press-fit pots (Kord Products Inc. Brampton, Ontario, Canada) in a walk-in Conviron growth chamber (Conviron Inc. Winnipeg, Manitoba, Canada). The photoperiod was 12 h light (140 μmol m−2 s−1, 21° C.) and 12 h dark (19° C.) and the relative humidity was 60%.
- Three weeks after sowing, the plants were treated with one of three watering regimes: ample water, moderate drought or severe drought. The moderate and severe drought designations are relative to the ample water treatment for our experiment. Plants were individually watered using a bottle-top volumetric dispenser. Plants subjected to ample watering had continually moist soil (c. 95% of the soil water-carrying capacity) and weekly water applications ranging from 55 to 170 ml depending on the plant age. Ample water-treated plants never wilted between waterings, showed no signs of waterlogging and were healthy in appearance. Severe drought-treated plants had soil which dried out completely between watering (c. 20% of the soil water-carrying capacity) and had weekly water applications ranging from 10 to 50 ml. Severe drought-treated plants displayed considerable wilting between waterings. Plants receiving the moderate drought treatment received approximately twice the volume of water applied to the severe drought-treated plants (c. 40% of the soil water-carrying capacity). Moderate drought-treated plants showed some turgor loss between watering, but to a lesser extent compared with the severe drought-treated plants. Water application was adjusted for treatment and plant age, and all plants within a treatment received the same amount of water. Relative water content measurements of 5-wk-old fully expanded leaves from three blocks showed no significant differences between the genotypes for any watering regime, indicating that the levels of drought stress were consistent across genotypes (data not shown). The average leaf relative water contents for each treatment were 76% for ample water, 65% for moderate drought and 61% for severe drought. These values are within the wide range of leaf relative water content values utilized for Arabidopsis drought stress in other published reports (Gigon et al., 2004; Rizhsky et al., 2004).
- Plants were arranged in a split-plot design. Three trays, each representing one water level, were clustered in a block, and 12 blocks were placed on separate shelves in the growth chamber. Genotypes were randomly assigned a position within a tray with two genotype replicates per tray. There were two replicate plants×8 genotypes×3 treatments×12 blocks for a total population of 576 plants. The transition from vegetative to reproductive growth was assessed by recording the flowering time (days from sowing), when the first open flower was visible, for each plant. Because of the large population size and the fact that flowering times were affected by treatment and genotype, each plant was individually harvested 4 wks. after the plant began to flower and the following variables were recorded: inflorescence height (cm), number of primary lateral branches and number of fruits plus any pistil which showed elongation or swelling. Excised rosettes were dried at 70° C. until a constant mass was achieved, and the dry mass was determined. For three blocks of plants, five fruits were harvested (two from the main inflorescence and three from the lateral branches), and the seed number per fruit was determined using a dissecting scope. Aborted or shriveled seeds were excluded from the seeds per fruit measurements. Seed production was estimated for each plant in the three blocks which had seeds per fruit measurements as (total fruit number×seeds per fruit). Relative fitness (total seed number×mean total seed number for water level) was calculated post hoc for the three blocks for which total seed production was determined.
- Experiment-wide variances for genotype means under ample water and drought stress were calculated using Minitab 15 (Minitab Inc. State College, Pa., USA). F test equal variance tests were performed in Minitab between mutant and wild-type trait variances from ample water and severe drought stress treatments. Eighty-four F tests were performed and the sequential Bonferroni correction was applied to keep a table-wide a of 0.05 (Rice, 1989).
- Multivariate and univariate analyses were performed using Proc GLM in SAS 9.1 (SAS Inc. Cary, N.C., USA). As plant mortality resulted in an unbalanced design, the two genotype×treatment replicates within a block were averaged to enable analysis by Proc GLM. Multivariate analysis of variance (MANOVA) was first performed to identify whether there were significant effects of genotype, treatment, and genotype×treatment interaction for a suite of reproduction-related traits, including rosette mass, inflorescence height, lateral branch number and fruit number. A second MANOVA was performed using data only from the three blocks for which the seed number per fruit and total seed production were calculated. The second MANOVA included the following response variables: rosette mass, inflorescence height, lateral branch number, fruit number, seed number per fruit and total seed production. Univariate ANOVAs were performed following the MANOVAs in order to determine which traits showed significant variation for phenotypic plasticity (genotype×treatment interaction). Flowering time was analyzed using ANOVA only. For both the multivariate and univariate analyses, the split-plot experimental design required that the whole-plot factor, treatment, be tested over the whole-plot random error term, treatment×block. Genotype and genotype×treatment interaction were tested over the residual error. Data and residuals were examined to ensure that all ANOVA assumptions were satisfied. Fruit number and total seed production required transformation (square root) in order to satisfy ANOVA assumptions of normality and stable variance. Square root transformation was used, because it is the recommended method for normalizing counted data (Kuehl, 2000) and was the most effective transformation for meeting the ANOVA assumptions. The sequential Bonferroni correction was applied to the univariate P values to minimize inflation of table-wide error from multiple tests (Rice, 1989).
- To determine whether G-protein mutants showed significantly different plasticities relative to Col, contrasts were performed using SAS 9.1 to test a priori-selected comparisons on all traits which had a significant genotype×water level interaction. Because two alleles of each mutant were studied, the contrasts were designed to simultaneously test the plasticity of Col against the plasticities of both mutant alleles of each gene. Combining alleles limited the inflation of a and, at the same time, increased the biological validity of the experiment: if the two mutant alleles of the same gene showed divergent responses, statistically significant differences with Col would probably not be detected. However, reaction norms were also examined individually to ensure that alleles of the same gene had similar plasticity responses.
- The following contrasts were performed for the univariate analyses: Col against both alleles combined for gpa1, agb1 and gcr1 mutants for ample water vs moderate drought and ample water vs severe drought. The double mutant gpa1-4agb1-2 was tested against Col, and against both alleles combined of gpa1 or agb1 for ample water vs moderate drought and ample water vs severe drought. The sequential Bonferroni correction was applied to adjust for the inflation of
type 1 error and to maintain a table-wide a of 0.05 (Rice, 1989). It has been suggested that the sequential Bonferroni correction can be overly stringent when applied to ecological experiments; therefore, we also applied biological reasoning when interpreting each contrast (Moran, 2003). Because relative fitness (Stanton & Thiede, 2005) was a post hoc addition to our analysis, relative fitness was analyzed independently from the other response variables. ANOVA was performed to test whether genotype, treatment and genotype by treatment interaction were significant. - Both MANOVAs showed that there is significant genotype×treatment interaction (Tables 1 and 2) for the reproduction-related traits (both P values were <0.0001). Significant genotype and treatment effects were also observed; however, the treatment effect for the seven variable MANOVA performed on only three blocks (Table 2) could not be estimated as a result of insufficient error degrees of freedom. The MANOVAs indicated that significant genetic variation exists for reproductive trait plasticity. Univariate ANOVAs were then performed in order to determine which specific traits showed significant genotype×treatment interaction. The results from the univariate ANOVAs are shown in Table 3. We found significant genotype×environment interactions for all traits, even after the sequential Bonferroni correction was applied to the P values. Least-squares means and standard errors are listed for all alleles and water levels in Table S1 (see Supporting Information). F tests of specific contrasts between any of the G-protein mutants and Col for ample water vs moderate drought and ample water vs severe drought revealed that, for rosette mass, number of lateral branches, total seed production and flowering time, there were no significant differences in genotype×water level interactions (plasticities) after the application of the sequential Bonferroni correction (Table 4). However, significant differences in plasticities were observed between Col and the G-protein mutants for a number of reproduction-related traits (Table 4). Although the contrasts were performed on pooled alleles, reaction norms for mutant alleles of the same gene were examined in cases in which the contrasts were significant; such examination confirmed that both alleles of each gene did indeed show similar responses for all plasticity traits discussed here (see, for example, Figs S4-S7; Supporting Information). Interestingly, agb1 mutants showed significantly reduced plasticities relative to Col for inflorescence height (
FIG. 3 ), fruit number (FIG. 4 ) and seeds per fruit (FIG. 5 ), suggesting that AGB1 functions as a plasticity gene, mediating phenotypic plasticity in the reproductive phase of plant growth in response to water availability. The plasticities of the gpa1-4 agb1-2 double mutant for the most part resembled those of the single agb1 mutants. The exception was the inflorescence height, for which agb1 mutants showed significantly reduced plasticity relative to the double mutant (FIG. 3 , Table 4). gpa1 mutants showed increased plasticity for inflorescence height relative to Col (FIG. 3 ). The double mutant showed reduced plasticity relative to gpa1 for inflorescence height (FIG. 3 ), indicating that the double mutant shows an intermediate phenotype. gcr1 mutants showed increased plasticity for square root fruit number relative to the wild-type (FIG. 4 ). - For total seed production, no significant differences were found in the plasticities of Col and any of the G-protein mutants following the sequential Bonferroni correction (Table 2). However, it should be noted that, before statistical correction, contrasts between Col and agb1 in ample water vs moderate drought (P=0.0027) and ample water vs severe drought (P=0.003) were significant. Given that two independent alleles of agb1 were used in the experiment and their reaction norms showed reduced plasticity for total seed production (
FIG. 6 ; see also Fig. S7, Supporting Information) relative to Col, and also that the sequential Bonferroni correction can be overly conservative (Moran, 2003), we feel confident that AGB1 also mediates plasticity for total seed production. All G-protein mutants showed reduced fitness, defined here as total seed production, under ample water conditions, but the agb1 mutants and the double mutant gpa1agb1 showed increased fitness under both moderate and severe drought stress relative to Col (FIG. 6 ). The relative fitness data also corroborate rank changing among the genotypes in different environments (FIG. 7 ). A significant genotype effect (P<0.0001) and significant genotype×treatment interaction (P=0.0009) were observed for relative fitness. Although all mutant genotypes showed reduced relative fitness relative to Col under ample water growth conditions, agb1 mutants and the double mutant gpa1-4agb1-2 showed increased relative fitness relative to Col under drought stress. - Phenotypic Variance of within x Treatment Trait Means
- The within-treatment, experiment-wide variances of the trait means for the mutants and wild-type for ample water and severe drought treatments are shown in Table S2 (see Supporting Information). Equal variance tests were performed to determine whether the mutants showed increased or decreased phenotypic variation relative to the wild-type within a particular environment (Table S3). After correcting for multiple F tests using the sequential Bonferroni correction, only one null hypothesis was rejected. agb1-2 mutants showed significantly reduced variance (P<0.0005) for seed number per fruit under ample water relative to the wild-type. However, this reduction in variance was not observed in the second agb1 allele, agb1-1 (P=0.168), which brings into question the biological relevance of this statistically significant observation. Overall, phenotypic variance for traits within a given environment was not impacted significantly by G-protein mutations.
- Our data show that mutation of the sole Gβ subunit of Arabidopsis, AGB1, results in pleiotropic effects on the extent of plasticity in response to water availability. agb1 mutants showed reduced plasticity for the number of fruits, inflorescence height, seed number per fruit and total seed production relative to Col. Interestingly, agb1 mutants showed increased seed production per fruit under drought stress relative to the wild-type, and reduced seed production per fruit relative to the wild-type under well-watered conditions. The reduced plasticity of agb1 mutants resulted in enhanced fitness under drought stress, but was maladaptive under well-watered conditions. This conclusion was also supported when relative fitness was assessed. All G-protein mutants, that showed reduced plasticity for reproductive traits, including agb1, showed lower relative fitness relative to Col under ample water conditions. Under drought stress, agb1 mutants and the double mutant showed increased relative fitness relative to Col. The relative fitness data support the conclusion that the significant genotype×treatment interaction observed for total seed production can be attributed to rank changing among the genotypes, and is not a consequence of changes in variance or the square root transformation. It has also been noted that differences in phenotypic variation can be a consequence of age- or size-dependent ontogenetic drift (McConnaughay & Coleman, 1999). It should be noted that we observed no significant differences in rosette growth rate (as determined by projected leaf area calculations) among the genotypes within a given water treatment (Figs S1-S3, see Supporting Information), suggesting that the significant variation for plasticity observed cannot be attributed to ontogenetic drift. These results suggest that AGB1 is a plasticity gene, as it contributes to the shape of the phenotypic response under the environments tested in our experiment.
- Multiple alleles have not been used frequently in mutant studies of phenotypic plasticity; one exception is the study by Galen et al. (2004). The use of two independent mutant alleles of agb1 in this study strengthens our conclusion that AGB1 functions as a plasticity gene, at least in the environments tested. These results suggest that it would be worthwhile to pursue additional experiments to determine the extent to which G proteins function in regulating phenotypic plasticity under different stresses and in natural environments, where other resource limitations might influence the extent of plasticity observed. It has been recognized that there may also be costs associated with plasticity under some circumstances (Callahan et al., 2005; Ghalambor et al., 2007; Van Buskirk & Steiner, 2009). Although additional research under different environments is required, plasticity costs may be illustrated under our conditions by the fact that, under water limitation, the least plastic genotype, agb1, showed greater fitness (by both absolute and relative measures) than the more plastic genotypes.
- AGB1 has been shown previously to function in inflorescence and fruit development. agb1-1 was originally isolated in a screen for erecta-like mutations, where it showed a slightly reduced inflorescence height and significantly shortened, blunt-tipped fruits relative to the wild-type, phenotypes which were later also observed in agb1-2 (Lease et al., 2001; Ullah et al., 2003). In addition, it was shown that AGB1 was expressed ubiquitously throughout the plant, but its expression was elevated in flowers and highest in fruits (Lease et al., 2001). The shortened fruit phenotype corresponds to our findings that agb1 shows reduced seeds per fruit. This short phenotype is specific to agb1 mutants and is not observed in gpa1, agg1 and agg2 mutants (Ullah et al., 2003; Trusov et al., 2008). Functional selectivity of the Gβ subunit has been reported for other G-protein-mediated responses, including necrotrophic pathogen resistance (Llorente et al., 2005; Trusov et al., 2006), sugar inhibition of seed germination and lateral root formation (Chen et al., 2006).
- According to the paradigm of G-protein signaling, activation of GPA1 results in a conformational change in Gα and the release of the Gβγ dimer, and signal propagation. As the reduced plasticity phenotype is present in agb1 mutants and in gpa1agb1 double mutants, but not in gpa1 mutants, we can conclude that Gβ is responsible for signal integration or transduction, resulting in wild-type plasticity for the number of fruits, seed number per fruit and total seed production. In classical G-protein signaling, the a subunit typically requires the β subunit, not only for trimer reassembly, but also for GPCR association. Therefore, GPA1 activation is eliminated by both gpa1 and agb1 mutations, but AGB1 activation is abolished only by the agb1 mutation. For inflorescence height, gpa1 and agb1 display opposite phenotypes (enhanced and reduced plasticity, respectively), which also suggests that AGB1 is responsible for mediating wild-type plasticity, as free AGB1 is active in gpa1 mutants (potentially more so than in the wild-type), but is absent from agb1 mutants. gcr1 mutants showed increased plasticity relative to the wild-type for fruit number and, although GCR1 might function in the mediation of plasticity for fruit number, there are probably additional unknown GPCRs that contribute to the perception and/or integration of environmental input signals with regard to the regulation of phenotypic plasticity.
- G-Protein Mutations do not Affect Significantly the Level of Phenotypic Variation within an Environment
- Within a given environment, genetically identical organisms can show divergent phenotypes as a result of stochasticity in gene expression. These random events among cellular molecules can modify cell status and consequently result in phenotypic changes at the organismal level (often thought of as experimental ‘noise’). Stochasticity is generally thought to be detrimental to fitness; however, it can also be a source of heterogeneity, which can provide a fitness benefit in fluctuating environments (Kaern et al., 2005; Raser & O'Shea, 2005). A G-protein mutation might increase or decrease the ability of a cell to buffer itself against stochasticity, and therefore it has been hypothesized that G-protein mutations might affect the amount of phenotypic variation within an environment (Assmann, 2004). To test this hypothesis, we compared the phenotypic variances (Table S2) of the G-protein mutants within an environment with the phenotypic variance of the wild-type within the same environment. We found that the within-environment phenotypic variation was not altered significantly by mutations in G-protein subunit genes (Table S3, Supporting Information), suggesting that this hypothesis is not supported: G-protein mutations do not affect the range of possible phenotypes within our environments.
- agb1 Mutants Show Enhanced Fitness Under Drought Stress
- Based on their previously reported altered stomatal sensitivities to ABA (Wang et al., 2001; Pandey & Assmann, 2004; Fan et al., 2008), it is possible to make predictions concerning the fitness benefits or costs that G-protein mutants may incur when grown under drought stress conditions. However, although gpa1 and agb1 are hyposensitive with regard to the ABA inhibition of stomatal opening, but show wild-type ABA promotion of stomatal closure (Wang et al., 2001; Fan et al., 2008), and gcr1 mutants are hypersensitive towards both ABA inhibition of stomatal opening and promotion of closure (Pandey & Assmann, 2004), our detailed phenotypic analysis of whole-plant traits under controlled water stress conditions did not support the predictions that would be made based on these stomatal response phenotypes. gcr1 mutants, despite their stomatal hypersensitivity to ABA and their reported improved recovery following drought stress (Pandey & Assmann, 2004), showed no fitness advantage relative to the wild-type under drought stress or well-watered conditions. Although we predicted that gpa1 and agb1 mutants would show reduced fitness under drought stress based on the partial ABA insensitivity of their stomatal phenotypes, agb1 mutants (but not gpa1 mutants) exhibited increased fitness under drought stress and reduced fitness under well-watered conditions. The pleiotropic nature of G-protein mutations may, in part, explain why the stomatal response phenotypes were not predictive of plant fitness under drought stress. Recently, it has been shown that G proteins regulate stomatal density, GPA1 as a positive modulator and AGB1 as a negative regulator (Zhang et al., 2008a), which may have implications for water loss and carbon assimilation under different environmental conditions. In addition, agb1 mutants and, to a lesser degree, gpa1 mutants are hypersensitive to ABA inhibition of germination and root growth (Pandey et al., 2006), which may contribute to survival under drought stress. Nevertheless, although the lack of plasticity of agb1 in reproductive-related traits resulted in a fitness advantage under drought stress, lack of plasticity was maladaptive for agb1 under well-watered conditions. Therefore, G proteins do contribute to plant fitness, in part via the regulation of phenotypic plasticity. Given that climate change can lead to increased variability in environments and resources, this finding could have important agronomical implications, as agb1 mutants show reduced plasticity and therefore more stable yields across environments.
- Phenotypic analysis of G-protein mutants under multiple environmental conditions has identified novel functions of plant heterotrimeric G proteins in the regulation of the phenotypic plasticity of inflorescence height, seed number per fruit, fruit number and total seed production. We also found that the known altered guard cell sensitivities towards ABA did not predict the fitness outcomes of the mutants under drought stress. All G-protein mutants studied showed reduced fitness under well-watered as well as drought environments, with the exception of agb1-containing mutants which showed improved fitness under drought stress conditions. These results thus attest to one of the apparent paradoxes of plant G-protein signaling: although G-protein mutation is not lethal, it does, in fact, result in nonoptimal phenotypes under some environmental conditions.
- G proteins mediate responses to ABA, auxin, brassinosteroids, gibberellins and sugars, environmental signals, including ozone and pathogens, and intrinsic, unknown developmental cues mediating leaf shape, stomatal density and fruit shape. A fundamental question in G-protein signaling is how only two heterotrimeric G-protein combinations (GPA1/AGB1/AGG1 and GPA1/AGB1/AGG2) can transduce such a diversity of signals. Our results, implicating heterotrimeric G proteins in the mediation of phenotypic plasticity responses, support the model that G proteins function, at least in part, as cross-talk hubs, integrating signals, rather than directly transducing them (Assmann, 2004), thereby tweaking a phenotype relative to the environment at hand. More studies are warranted in which G-protein regulation of plasticity is examined across additional environmental gradients, as well as across generations, in order to further elucidate this novel contribution of heterotrimeric G proteins to plant development and fitness. It will be interesting to assess the extent to which the results presented here would extrapolate to field studies: studies in highly controlled environments are important steps towards the design of such experiments. Although QTL-based studies have supported the existence of plasticity genes, the present study is one of a few that has directly tested the regulation of phenotypic plasticity by specific genes. Additional plasticity studies with other environmental signaling mutants will be integral to our understanding of whether plasticity genes are rare or common in plant and other genomes, and to gaining an insight into the genetic basis of phenotypic plasticity.
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TABLE 1 Wilks' lambda, F and P values from multivariate analysis of variance (MANOVA) including the following response variables: rosette mass, inflorescence height, number of lateral branches and square root number of fruits; data from all 12 experimental blocks Wilks' Denominator Effect lambda F value Numerator d.f. d.f. P value Genotype 0.0419 41.48 28 823.49 <0.0001 Treatment 0.0056 58.65 8 38 <0.0001 Genotype × 0.2046 8 56 889.04 <0.0001 treatment -
TABLE 2 Wilks' lambda, F and P values from multivariate analysis of variance (MANOVA) including the following response variables: rosette mass, inflorescence height, number of lateral branches and square root number of fruits; seed number per fruit and square root total seed production; data from the three experimental blocks for which seed number per fruit was obtained Wilks' Numerator Denominator Effect lambda F value d.f. d.f. P value Genotype 0.00531 8.651 421 1771 <0.00011 Treatment Genotype × 0.0208 2.54 84 212.6 <0.0001 treatment 1Multivariate analysis of the significance of the treatment effect could not be performed because of insufficient error d.f. (treatment × block is the appropriate error term given the split-plot experimental design). -
TABLE 3 F and (P values) for all fixed effects for univariate analyses of variance (ANOVAs); treatment was tested over the treatment × block error term, and geotype and genotype × treatment were tested over the residual error; all P values were significant before and after sequential Bonferroni correction Effect Genotype × Response Variable Genotype Treatment treatment Rosette mass 28.08 (≦0.0001) 744.91 (≦0.0001) 8.43 (≦0.0001) Inflorescence height 40.89 (≦0.0001) 541.7 (≦0.0001) 12.79 (≦0.0001) Number of lateral branches 108.9 (<0.0001) 272.7 (≦0.0001) 12.79 (≦0.0001) Square root number of fruits 9.35 (≦0.0001) 320.91 (≦0.0001) 13 (≦0.0001) Seeds per fruit1 10.47 (≦0.0001) 256.7 (≦0.0001) 4.23 (≦0.0001) Seed production1 7.79 (≦0.0001) 220.07 (≦0.0001) 2.88 (≦0.0041) Flowering time 50.37 (≦0.0001) 12.7117323 (0.0002) 3.23 (0.0001) 1Only three blocks were used to assess seeds per fruit and seed production. -
TABLE 4 P values of univariate contrasts on genotype x treatment (ample, ample water, moderate, moderate drought severe, severe drought) Total Inflores- No. Seeds seed Flower- Rosette cence lateral Sq. rt. per pro- ing Response variable mass height branches no. fruit fruit1 duction1 Time Contrast Col vs both agb1 ample 0.0137 0.0001 0.104 ≦ 0.0001 0.0003 0.0027 0.9828 vs moderate Col vs both agb1 ample 0.002 0.0022 0.0183 0.0091 ≦ 0.0001 0.003 0.645 vs severe Col vs double ample vs 0.0017 0.472 0.0876 ≦ 0.0001 0.0052 0.0323 0.8351 moderate Col vs double ample vs 0.002 0.2793 0.1045 0.1331 0.0049 0.0715 0.3148 severe Col vs both gcr1 ample 0.9661 0.8617 0.882 0.0024 0.7164 0.7598 0.5887 vs moderate Col vs both gcr1 ample 0.2308 0.1975 0.4888 ≦ 0.0001 0.1723 0.8404 0.0859 vs severe Col vs both gpa1 ample 0.3071 0.0007 0.1035 0.7262 0.5187 0.8318 0.496 vs moderate Col vs both gpa1 ample 0.0301 ≦ 0.0001 0.0844 0.2792 0.1083 0.2709 0.0019 vs severe Double vs both agb1 0.2399 0.0024 0.7284 0.5487 0.5548 0.5354 0.8268 ample vs moderate Double vs both agb1 0.6283 ≦ 0.0001 0.6209 0.3752 0.3024 0.3139 0.1056 ample vs severe Double vs both gpa1 ≦ 0.0001 ≦ 0.0001 0.7291 ≦ 0.0001 ≦ 0.0002 0.0083 0.6594 ample vs moderate Double vs both gpa1 ≦ 0.0001 ≦ 0.0001 0.882 0.0051 0.0812 0.3138 0.0483 ample vs severe 1Data from three blocks only. Values in bold were significant before application of the sequential Bonferroni correction; values in italic and with underlines were significant after application of the Bonferroni correction. -
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- Genbank RGA1 cDNA sequence:
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RGA1 coding region sequence: SEQ ID NO: 1 ATGGGCTCATCCTGTAGCAGATCTCATTCTTTAAGTGAGGCTGAA ACAACCAAAAATGCAAAATCTGCAGACATTGACAGGCGAATTTTG CAAGAGACAAAAGCAGAGCAACACATCCACAAGCTCTTACTTCTT GGTGCGGGAGAATCAGGGAAGTCTACGATATTTAAACAGATTAAG CTCCTTTTCCAAACTGGCTTTGATGAGGCAGAACTTAGGAGCTAC ACATCAGTTATCCATGCAAACGTCTATCAGACAATTAAAATACTA TATGAAGGAGCAAAAGAACTCTCACAAGTGGAATCAGATTCCTCA AAGTATGTTATATCCCCAGATAACCAGGAAATTGGAGAAAAACTA TCAGATATTGATGGCAGGTTGGATTATCCACTGCTGAACAAAGAA CTTGTACTCGATGTAAAAAGGTTATGGCAAGACCCAGCCATTCAG GAAACTTACTTACGTGGAAGTATTCTGCAACTTCCTGATTGTGCA CAATACTTCATGGAAAATTTGGATCGATTAGCTGAAGCAGGTTAT GTGCCAACAAAGGAGGATGTGCTTTATGCAAGAGTACGGACAAAT GGTGTTGTACAAATACAATTTAGTCCTGTTGGAGAAAACAAAAGA GGTGGAGAGGTATATAGGTTGTATGATGTAGGAGGCCAGAGGAAT GAGAGGAGAAAGTGGATTCATCTTTTTGAAGGTGTTAATGCGGTA ATCTTTTGTGCTGCCATTAGCGAATATGATCAGATGCTATTTGAA GATGAGACAAAAAACAGAATGATGGAGACCAAGGAACTCTTTGAC TGGGTTTTAAAGCAAAGATGTTTTGAGAAAACATCATTCATTCTG TTTCTCAACAAATTTGATATATTCGAGAAGAAAATACAAAAGGTT CCTTTAAGTGTGTGCGAGTGGTTTAAAGACTACCAGCCTATTGCA CCTGGGAAACAGGAGGTTGAACATGCATATGAGTTTGTCAAGAAG AAGTTTGAAGAGCTCTACTTCCAGAGCAGCAAGCCTGACCGTGTG GACCGCGTCTTCAAAATCTACAGAACTACGGCCCTAGACCAGAAA CTTGTAAAGAAGACATTCAAGTTGATTGATGAGAGCATGAGACGC TCCAGGGAAGGAACTTGA d1 coding region sequence (from our cDNA sequencing) SEQ ID NO 2ATGGGCTCATCCTGTAGCAGATCTCATTCTTTAAGTGAGGCTGAA ACAACCAAAAATGCAAAATCTGCAGACATTGACAGGCGAATTTTG CAAGAGACAAAAGCAGAGCAACACATCCACAAGCTCTTACTTCTT GGTGCGGGAGAATCAGGGAAGTCTACGATATTTAAACAGATTAAG CTCCTTTTCCAAACTGGCTTTGATGAGGCAGAACTTAGGAGCTAC ACATCAGTTATCCATGCAAACGTCTATCAGACAATTAAAATACTA TATGAAGGAGCAAAAGAACTCTCACAAGTGGAATCAGATTCCTCA AAGTATGTTATATCCCCAGATAACCAGGAAATTGGAGAAAAACTA TCAGATATTGATGGCAGGTTGGATTATCCACTGCTGAACAAAGAA CTTGTACTCGATGTAAAAAGGTTATGGCAAGACCCAGCCATTCAG GAAACTTACTTACGTGGAAGTATTCTGCAACTTCCTGATTGTGCA CAATACTTCATGGAAAATTTGGATCGATTAGCTGAAGCAGGTTAT GTGCCAACAAAGGAGGATGTGCTTTATGCAAGAGTACGGACAAAT GGTGTTGTACAAATACAATTTAGTCCTGTTGGAGAAAACAAAAGA GGTGGAGAGGTATATAGGTTGTATGATGTAGGAGGCCAGAGGAAT GAGAGGAGAAAGTGGATTCATCTTTTTGAAGGTGTTAATGCGGTA ATCTTTTGTGCTGCCATTAGCGAATATGATCAGATGCTATTTGAA GATGAGACAAAAAACAGAATGATGGAGACCAAGGAACTCTTTGAC TGGGTTTTAAAGCAAAGATGTTTTGAGAAAACATCATTCATTCTG TTTCTCAACAAATTTGATATATTCGAGAAGAAAATACAAAAGGTT CCTTTAAGTGTGCGAGTGGTTTAAAGACTACCAGCCTATTGCACC TGGGAAACAGGAGGTTGAACATGCATATGAGTTTGTCAAGAAGAA GTTTGAAGAGCTCTACTTCCAGAGCAGCAAGCCTGACCGTGTGGA CCGCGTCTTCAAAATCTACAGAACTACGGCCCTAGACCAGAAACT TGTAAAGAAGACATTCAAGTTGATTGATGAGAGCATGAGACGCTC CAGGGAAGGAACTTGA
Genbank RGA1 protein sequence: -
-
RGA1 protein sequence: SEQ ID NO: 3 MGSSCSRSHSLSEAETTKNAKSADIDRRILQETKAEQHIHKLLLL GAGESGKSTIFKQIKLLFQTGFDEAELRSYTSVIHANVYQTIKIL YEGAKELSQVESDSSKYVISPDNQEIGEKLSDIDGRLDYPLLNKE LVLDVKRLWQDPAIQETYLRGSILQLPDCAQYFMENLDRLAEAGY VPTKEDVLYARVRTNGVVQIQFSPVGENKRGGEVYRLYDVGGQRN ERRKWIHLFEGVNAVIFCAAISEYDQMLFEDETKNRMMETKELFD WVLKQRCFEKTSFILFLNKFDIFEKKIQKVPLSVCEWFKDYQPIA PGKQEVEHAYEFVKKKFEELYFQSSKPDRVDRVFKIYRTTALDQK LVKKTFKLIDESMRRSREGT- d1 predicted protein sequence (based on our cDNA sequencing) SEQ ID NO: 4 MGSSCSRSHSLSEAETTKNAKSADIDRRILQETKAEQHIHKLLLL GAGESGKSTIFKQIKLLFQTGFDEAELRSYTSVIHANVYQTIKIL YEGAKELSQVESDSSKYVISPDNQEIGEKLSDIDGRLDYPLLNKE LVLDVKRLWQDPAIQETYLRGSILQLPDCAQYFMENLDRLAEAGY VPTKEDVLYARVRTNGVVQIQFSPVGENKRGGEVYRLYDVGGQRN ERRKWIHLFEGVNAVIFCAAISEYDQMLFEDETKNRMMETKELFD WVLKQRCFEKTSFILFLNKFDIFEKKIQKVPLSVRVV-
MSU Rice Genome Annotation Project LOC_Os05g26890 (RGA1) genomic sequence (Oryza sativa ssp japonica cv. Nipponbare—Sequence Release 7)
Sequence aligns 100% with the RGA1 sequence found within genbank clones:
Chromosome 5 clone OSJNBa0049D13 (accession AC144739)
Chromosome 5 clone OJ1005_D04 (accession AC117264) -
RGA1 genomic sequence: SEQ ID NO: 5 ATGGGCTCATCCTGTAGCAGATCTCATTCTTTAAGTGAGGCTGAA ACAACCAAAAATGCAAAAGTAAGTTAGCACTCGGACTTATTGAAC AAGTAAATGCTAACTCAATTCTTGATTTGAGAGTTGCCACATTTG GTTTCTTCTAATTCAGCTGGTAACAGTCTGCAGACATTGACAGGC GAATTTTGCAAGAGACAAAAGCAGAGCAACACATCCACAAGCTCT TACTTCTTGGTATTGCTAACTTTCCCAAATTTAAGTGGTCATTTT CCTTGTCACAATTATCTGTGCTACCTTTAGTATCTATTGGTTCAG AAAATTAATTGTTTATGTTGTTCCTATTTACCTCTATAAAAAAAC CTTTCTCATGTTATTTCCAAAAAAAAAGAAGATAAATAAATGTAT CCTAGAAATTTTTAGTTTGAACTTGTTCTCAATGTGGATCCATCC TTCTTTCTCTCTCTCAATTGCTTCTGTTTTAAGGTGCGGGAGAAT CAGGGAAGTCTACGATATTTAAACAGGTGATGAATGTTATATTCC ATGGAGAATCATAATCCGTACGCCGCTAGTTAGTCTGATGTATTC TTACTGTTCACCTGCAGATTAAGCTCCTTTTCCAAACTGGCTTTG ATGAGGCAGAACTTAGGAGCTACACATCAGTTATCCATGCAAACG TCTATCAGACAATTAAAGTATGCAATACTGGAAAGGGTGTGTCTT TTTTTTCTTATTGCAAAGTGGGGATTATGTAGGAGATTCGACTAG GGATTTGTATTCTGTTCATAAGGAAATGCGTTCATACTTTTCCTT TTTGTCGAGTAATGTGTTAAATGTTAACAGATACTATATGAAGGA GCAAAAGAACTCTCACAAGTGGAATCAGATTCCTCAAAGTATGTT ATATCCCCAGATAACCAGGTTTGTGCTTACTCTTTACTCAACAGT TAAAGCTAAATCTGTGCATATGAACATGTCTTGTTAAATCTGGGA ATACAAACATTTTGATTTGCAACATTTCTGTTGTAGTCAAGCTGC TCGGCTCTATGTTTTAACCTGTTAAGACCTTGTAGACTGTGCTCG GCTCTATTGTAGTCTTATATTTTACACGGTCATTCTATAATGAAA ACTTGAAAAAGATATCTATTGAACCGTACAATGTACTGAACAAAG TAGAAAAGAACAATGAGATTTTGTAACATTTATTCTTCCTTGTTT ATTTGATTGCTTCAGACAATTGTTGATATGCTAAAAATAACTTGG TATCAAATGTGGGTGTTATAAGATTCAATTTTTTTCTCAACCAGG TTAAAAAAAGTATACCTTTGTGCATTTACCTTGTTCCGTTGCTTT GGAACTTTAAAGGAAAACTGACTTTTCTTAGGCATTGAAAGACAA ATATCACCAGTTTCACACTGTACACCTTACCAACCAATTTTGTTT CTTAGATGTCATTTACTTTGTCATATCATCAGGAAATTGGAGAAA AACTATCAGATATTGATGGCAGGTTGGATTATCCACTGCTGAACA AAGAACTTGTACTCGATGTAAAAAGGTTATGGCAAGACCCAGCCA TTCAGGTGAAAACAAATAGCCATTCAAATCTTTTGAAGTTATATA GTTTTCCTGGCCAGGTGTGCTGAAGCAATGCTCTATACTGTAGGA AACTTACTTACGTGGAAGTATTCTGCAACTTCCTGATTGTGCACA ATACTTCATGGAAAATTTGGATCGATTAGCTGAAGCAGGTTATGT GCCAACAAAGGTGTGCTGTCCATGTTCATAGACAATTATTTACAT ATTCTCAGATATTTGTGCTGACACCATTTCATGTTGATTTTTAGT CTACTTAGTCAGAGGTTGTCAAATGGTTAACTATGTGTACTGAGT CAGAGGTTGCCAAATAGTTTTAAAAGATGGGCATATGTTTATCCT TATCTTTTAAATAATATTGGAGGCTATCCTTTAAAATTCAATATT AGGGAGGAGAAACTATTATTCTACCGTTATTACGCAGTCTACATA ACGAAGGTAAAAAATGTCCCTGTGAAACATAGGGTGCAAAACTGC TGTGAATAAAACTCTACTTATCTAAGCACCTTGAGCTTTTGAGTT CCCACATATTAATCTTATGACACTAGCATATATTTTTTTTGTTCA GTTCCTTCAATAAGTTGCAAACCACAAATATGATCACTGTACCAT CCACTTTTGCAACCATTTCCCGTCATTTCTTAAGCATAGAAAATT GTTTGTCACTTGTTTAAGTCCACACTGCATCAAAATTCCAATTAA CATTGTGTGTGCTAAGTGAAGATATGACTCCATATTTCTGCATTT AGCAGTCTGATGGATAATTTGTGATTGTACCTTGTCTAATGGTTC GTTTGAAAGGCTGGTAGTTGATCTTCCATACTTAAGAATGCTTGC AGTATTATAGTTGTCAATATTATGAGTCATTTTGCAGGAGGATGT GCTTTATGCAAGAGTACGGACAAATGGTGTTGTACAAATACAATT TAGGTAATCTGCTGACACTATTTTTTGCACATTTTTTTGCTGGTT GCTCTACTATGTACAGAACGACAAGTTGAAGTCCTTTTTTTCTCC CCTTTCACTTCTAAGATATGACCTGAGAGGTTCTGAATGTAGCTG TTTTAAGATGAGTTGAATCATCTAGTTAACTGGGTTTCTTTCTGC AGTCCTGTTGGAGAAAACAAAAGAGGTGGAGAGGTATATAGGTTG TATGATGTAGGAGGCCAGAGGAATGAGAGGAGAAAGTGGATTCAT CTTTTTGAAGGTGTTAATGCGGTAATCTTTTGTGCTGCCATTAGC GAGTAAGTACAATTTTTTTGATTGTTGAACTTATCCTAATCTGCT AAGTTCTTCTCATAGGCTTCTTGTTCATTTCAGATATGATCAGAT GCTATTTGAAGATGAGACAAAAAACAGAATGATGGAGACCAAGGA ACTCTTTGACTGGGTTTTAAAGCAAAGATGTTTTGAGGTCTGCAT GCATCCATCTCTGCAACCTTTGTGCTCATGCTTTTTTTCTCATTT TGAAACTAATTACGGTGCTATATTGACCATCAGAAAACATCATTC ATTCTGTTTCTCAACAAATTTGATATATTCGAGAAGAAAATACAA AAGGTAAGGCCTGCTCTTTGTACCAATGCATAGTTTAGTACTAAA TGTTACCAACATTTATGTTTACGCTGGTTACGTAGGTTCCTTTAA GTGTGTGCGAGTGGTTTAAAGACTACCAGCCTATTGCACCTGGGA AACAGGAGGTTGAACATGCATATGAGTGAGTGCACTACTCGCCCT CTCAGATGAACATGGGCATTTGGCCATTTGTAATGTTGCTGCATG GTGCACTTATATGCCTTGATAAGTTTTTCCATTCTAATGTTATAT AGTATCAAACGTTCATCATTACTGTGGCTTATGGTCTGGAGTGAC GTTTTACAGGTTTGTCAAGAAGAAGTTTGAAGAGCTCTACTTCCA GAGCAGCAAGCCTGACCGTGTGGACCGCGTCTTCAAAATCTACAG AACTACGGCCCTAGACCAGAAACTTGTAAAGAAGACATTCAAGTT GATTGATGAGAGCATGAGACGCTCCAGGGAAGGAACTTGA d1 allele sequence (from our genomic DNA sequencing) SEQ ID NO: 6 ATGGGCTCATCCTGTAGCAGATCTCATTCTTTAAGTGAGGCTGAA ACAACCAAAAATGCAAAAGTAAGTTAGCACTCGGACTTATTGAAC AAGTAAATGCTAACTCAATTCTTGATTTGAGAGTTGCCACATTTG GTTTCTTCTAATTCAGCTGGTAACAGTCTGCAGACATTGACAGGC GAATTTTGCAAGAGACAAAAGCAGAGCAACACATCCACAAGCTCT TACTTCTTGGTATTGCTAACTTTCCCAAATTTAAGTGGTCATTTT CCTTGTCACAATTATCTGTGCTACCTTTAGTATCTATTGGTTCAG AAAATTAATTGTTTATGTTGTTCCTATTTACCTCTATAAAAAAAC CTTTCTCATGTTATTTCCAAAAAAAAAGAAGATAAATAAATGTAT CCTAGAAATTTTTAGTTTGAACTTGTTCTCAATGTGGATCCATCC TTCTTTCTCTCTCTCAATTGCTTCTGTTTTAAGGTGCGGGAGAAT CAGGGAAGTCTACGATATTTAAACAGGTGATGAATGTTATATTCC ATGGAGAATCATAATCCGTACGCCGCTAGTTAGTCTGATGTATTC TTACTGTTCACCTGCAGATTAAGCTCCTTTTCCAAACTGGCTTTG ATGAGGCAGAACTTAGGAGCTACACATCAGTTATCCATGCAAACG TCTATCAGACAATTAAAGTATGCAATACTGGAAAGGGTGTGTCTT TTTTTTCTTATTGCAAAGTGGGGATTATGTAGGAGATTCGACTAG GGATTTGTATTCTGTTCATAAGGAAATGCGTTCATACTTTTCCTT TTTGTCGAGTAATGTGTTAAATGTTAACAGATACTATATGAAGGA GCAAAAGAACTCTCACAAGTGGAATCAGATTCCTCAAAGTATGTT ATATCCCCAGATAACCAGGTTTGTGCTTACTCTTTACTCAACAGT TAAAGCTAAATCTGTGCATATGAACATGTCTTGTTAAATCTGGGA ATACAAACATTTTGATTTGCAACATTTCTGTTGTAGTCAAGCTGC TCGGCTCTATGTTTTAACCTGTTAAGACCTTGTAGACTGTGCTCG GCTCTATTGTAGTCTTATGTTTTACACGGTCATTCTATAATGAAA ACTTGAAAAAGATATCTATTGAACCGTACAATGTACTGAACAAAG TAGAAAAGAACAATGAGATTTTGTAACATTTATTCTTCCTTGTTT ATTTGATTGCTTCAGACAATTGTTGATATGCTAAAAATAACTTGG TATCAAATGTGGGTGTTATAAGATTCAATTTTTTTCTCAACCAGG TTAAAAAAAGTATACCTTTGTGCATTTACCTTGTTCCGTTGCTTT GGAACTTTAAAGGAAAACTGACTTTTCTTAGGCATTGAAAGACAA ATATCACCAGTTTCACACTGTACACCTTACCAACCAATTTTGTTT CTTAGATGTCATTTACTTTGTCATATCATCAGGAAATTGGAGAAA AACTATCAGATATTGATGGCAGGTTGGATTATCCACTGCTGAACA AAGAACTTGTACTCGATGTAAAAAGGTTATGGCAAGACCCAGCCA TTCAGGTGAAAACAAATAGCCATTCAAATCTTTTGAAGTTATATA GTTTTCCTGGCCAGGTGTGCTGAAGCAATGCTCTATACTGTAGGA AACTTACTTACGTGGAAGTATTCTGCAACTTCCTGATTGTGCACA ATACTTCATGGAAAATTTGGATCGATTAGCTGAAGCAGGTTATGT GCCAACAAAGGTGTGCTGTCCATGTTCATAGACAATTATTTACAT ATTCTCAGATATTTGTGCTGACACCATTTCATGTTGATTTTTAGT CTACTTAGTCAGAGGTTGTCAAATGGTTAACTATGTGTACTGAGT CAGAGGTTGCCAAATAGTTTTAAAAGATGGGCATATGTTTATCCT TATCTTTTAAATAATATTGGAGGCTATCCTTTAAAATTCAATATT AGGGAGGAGAAACTATTATTCTACCGTTATTACGCAGTCTACATA ACGAAGGTAAAAAATGTCCCTGTGAAACATAGGGTGCAAAACTGC TGTGAATAAAACTCTACTTATCTAAGCACCTTGAGCTTTTGAGTT CCCACATATTAATCTTATGACACTAGCATATATTTTTTTTGTTCA GTTCCTTCAATAAGTTGCAAACCACAAATATGATCACTGTACCAT CCACTTTTGCAACCATTTCCCGTCATTTCTTAAGCATAGAAAATT GTTTGTCACTTGTTTAAGTCCACACTGCATCAAAATTCCAATTAA CATTGTGTGTGCTAAGTGAAGATATGACTCCATATTTCTGCATTT AGCAGTCTGATGGATAATTTGTGATTGTACCTTGTCTAATGGTTC GTTTGAAAGGCTGGTAGTTGATCTTCCATACTTAAGAATGCTTGC AGTATTATAGTTGTCAATATTATGAGTCATTTTGCAGGAGGATGT GCTTTATGCAAGAGTACGGACAAATGGTGTTGTACAAATACAATT TAGGTAATCTGCTGACACTATTTTTTGCACATTTTTTTGCTGGTT GCTCTACTATGTACAGAACGACAAGTTGAAGTCCTTTTTTTCTCC CCTTTCACTTCTAAGATATGACCTGAGAGGTTCTGAATGTAGCTG TTTTAAGATGAGTTGAATCATCTAGTTAACTGGGTTTCTTTCTGC AGTCCTGTTGGAGAAAACAAAAGAGGTGGAGAGGTATATAGGTTG TATGATGTAGGAGGCCAGAGGAATGAGAGGAGAAAGTGGATTCAT CTTTTTGAAGGTGTTAATGCGGTAATCTTTTGTGCTGCCATTAGC GAGTAAGTACAATTTTTTTGATTGTTGAACTTATCCTAATCTGCT AAGTTCTTCTCATAGGCTTCTTGTTCATTTCAGATATGATCAGAT GCTATTTGAAGATGAGACAAAAAACAGAATGATGGAGACCAAGGA ACTCTTTGACTGGGTTTTAAAGCAAAGATGTTTTGAGGTCTGCAT GCATCCATCTCTGCAACCTTTGTGCTCATGCTTTTTTTCTCATTT TGAAACTAATTACGGTGCTATATTGACCATCAGAAAACATCATTC ATTCTGTTTCTCAACAAATTTGATATATTCGAGAAGAAAATACAA AAGGTAAGGCCTGCTCTTTGTACCAATGCATAGTTTAGTACTAAA TGTTACCAACATTTATGTTTACGCTGGTTACGTAGGTTCCTTTAA GTGTGCGAGTGGTTTAAAGACTACCAGCCTATTGCACCTGGGAAA CAGGAGGTTGAACATGCATATGAGTGAGTGCACTACTCGCCCTCT CAGATGAACATGGGCATTTGGCCATTTGTAATGTTGCTGCATGGT GCACTTATATGCCTTGATAAGTTTTTCCATTCTAATGTTATATAG TATCAAACGTTCATCATTACTGTGGCTTATGGTCTGGAGTGACGT TTTACAGGTTTGTCAAGAAGAAGTTTGAAGAGCTCTACTTCCAGA GCAGCAAGCCTGACCGTGTGGACCGCGTCTTCAAAATCTACAGAA CTACGGCCCTAGACCAGAAACTTGTAAAGAAGACATTCAAGTTGA TTGATGAGAGCATGAGACGCTCCAGGGAAGGAACTTGA
Other than the expected 2 bp deletion at 3245 and 3246, the substitution at 1099 (located in the middle of an intron) is the only polymorphism found between the MSU and our d1 sequences.
Claims (20)
1. A genetically modified plant with improved seed production in drought conditions and/or improved survival at high planting densities compared to the seed production of a corresponding plant with no such modification; said modified plant having reduced G-protein activity when compared to a non-modified plant in the same conditions, said G-protein selected from the group consisting of:
Gα, Gβ, Gγ, GPCR, and GCR1.
2. The modified plant of claim 1 wherein said G-protein protein activity is created by introducing an inhibition construct to said plant or an ancestor thereof.
3. Seed of the plant of claim 1 .
4. The plant of claim 1 wherein said plant is a maize, cotton, soybean, wheat, barley, rice, rye, oat or sorghum plant.
5. The plant of claim 1 wherein said plant is a rice plant.
6. The plant of claim 1 wherein said G-protein is a G-protein alpha subunit.
7. The plant of claim 1 wherein said modification is a loss of function modification.
8. The plant of claim 1 wherein said plant includes a heterologous construct that inhibits expression of a G-protein alpha subunit.
9. The plant of claim 1 wherein said plant includes a G-protein alpha subunit expression cassette that is an inhibitory cassette.
10. The plant of claim 1 wherein said G-protein alpha subunit sequence is SEQ ID NO: 1 or 5.
11. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence set forth in SEQ ID NO:1, or SEQ ID NO:3;
(b) a nucleotide sequence which encodes the amino acid sequence of SEQ ID NO:2 or 4;
(c) a nucleotide sequence which includes about 90% identity to the full length sequence of SEQ I NO:1 or 3.
12. A nucleic acid construct comprising a nucleic acid molecule of claim 11 , wherein the nucleic acid is operably linked to a promoter that drives transcription in a plant cell.
13. A plant comprising in its genome at least one nucleic acid molecule of claim.
14. Seed of the plant of claim 13 .
15. The nucleic acid construct of claim 12 wherein said construct is an inhibition construct.
16. A method for conferring or improving seed production under drought conditions and/or improved in a rice plant, said method comprising:
(a) transforming said plant with a nucleic acid molecule comprising an inhibition construct with a heterologous sequence operably linked to a promoter that induces transcription of said heterologous sequence in a plant cell; and
(b) regenerating stably transformed plants, wherein said heterologous sequence comprises an inhibition nucleic acid molecule that reduces the activity or expression of one or more G-protein encoding sequences.
17. The method of claim 16 further comprising obtaining genetically modified progeny plants of one or more generations with increased growth in drought conditions.
18. The method of claim 16 wherein the inhibition construct is by co-suppression, antisense suppression, or RNAi-mediated suppression.
19. The method of claim 16 wherein the decrease is obtained by introduction of an inhibitory sequence.
20. The method of claim 17 wherein the progeny plants are obtained by selfing, crossing with another plant or clonal propagation.
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US13/866,401 US9434957B2 (en) | 2012-06-28 | 2013-04-19 | Methods of improving drought tolerance and seed production in rice |
US15/246,932 US20160362703A1 (en) | 2012-06-28 | 2016-08-25 | Methods of improving drought tolerance and seed production in rice |
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CN108967181A (en) * | 2018-07-18 | 2018-12-11 | 广东省农业科学院水稻研究所 | A kind of method for creating of Rice Germplasm Resources and the application in the rice production of hybrid seeds |
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CN110468150B (en) * | 2019-08-21 | 2020-10-30 | 浙江大学 | Application of RGS1 gene as negative regulatory factor in improving tomato bacterial leaf spot resistance in low-irradiation environment |
US11612110B2 (en) * | 2020-11-04 | 2023-03-28 | Fluence Bioengineering, Inc. | Environmental parameters for growing crops under high intensity lighting |
CN114874300B (en) * | 2021-02-23 | 2023-07-14 | 中国农业科学院作物科学研究所 | TaDRS1 protein and application of encoding gene thereof in regulation and control of wheat plant height and grain shape |
CN115005034B (en) * | 2022-06-14 | 2023-12-08 | 江苏艾津作物科技集团有限公司 | Device and method for identifying stress resistance of exogenous biological stimulus to rice seedling stage |
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CN108967181A (en) * | 2018-07-18 | 2018-12-11 | 广东省农业科学院水稻研究所 | A kind of method for creating of Rice Germplasm Resources and the application in the rice production of hybrid seeds |
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